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Biosynthetic pathway to neuromelanin andits aging processKazumasa Wakamatsu, Takaya Murase, Fabio A. Zucca,

Luigi Zecca and Shosuke Ito

Submit your next paper to PCMR online at http://mc.manuscriptcentral.com/pcmr

DOI: 10.1111/pcmr.12014Volume 25, Issue 6, Pages 792-803

Biosynthetic pathway to neuromelanin and its agingprocessKazumasa Wakamatsu1, Takaya Murase1, Fabio A. Zucca2, Luigi Zecca2 and Shosuke Ito1

1 Department of Chemistry, Fujita Health University School of Health Sciences, Toyoake, Japan 2 Institute ofBiomedical Technologies – National Research Council of Italy, Segrate, Milano, Italy

CORRESPONDENCE Kazumasa Wakamatsu, e-mail: kwaka@fujita-hu.ac.jp

KEYWORDS 4-amino-3-hydroxyphenylethylamine/cysteine/dopamine/neuromelanin/thiazole-2,4,5-tri-carboxylic acid

PUBLICATION DATA Received 9 July 2012, revisedand accepted for publication 27 August 2012,published online 31 August 2012

doi: 10.1111/pcmr.12014

Summary

Using model compounds of the melanic component of neuromelanin (NM) prepared by tyrosinase oxidation at

various ratios of dopamine (DA) and cysteine (Cys) under physiological conditions, we examined a biosynthetic

pathway to NM and its aging process by following the time course of oxidation to NM and the subsequent

structural modification of NM under various heating conditions. Chemical degradation methods were applied to

the synthetic NM. 4-Amino-3-hydroxyphenylethylamine (4-AHPEA) and thiazole-2,4,5-tricarboxylic acid (TTCA)

were used as markers of benzothiazine and benzothiazole units, respectively. By following the time course of the

biosynthetic pathway of synthetic NM, we found that neurotoxic molecules are trapped in NM. An aging

simulation of synthetic NM showed that benzothiazine units in NM are gradually converted to benzothiazole

during the aging process. Thus, natural NM was found to be similar to aged (heated) NM prepared from a 2:1

molar ratio of DA and Cys.

Introduction

There are two chemically distinct types of melanin, that

is, black to brown eumelanin and yellow to reddish brown

pheomelanin (Ito and Wakamatsu, 2003, 2006; Simon and

Peles, 2010). Eumelanin is composed of oligomers of 5,6-

dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carbox-

ylic acid (DHICA), while pheomelanin is derived from

benzothiazine and benzothiazole units. Both types of

melanin pigment arise from tyrosinase oxidation of the

common precursor, L-tyrosine. L-Cysteine (Cys) is specif-

ically required for the production of pheomelanin.

Although much is known about melanins outside the

central nervous system, many basic questions remain to

be answered about melanins present in the brain.

Neuromelanin (NM) is composed of black to brown

pigments found mainly in the substantia nigra (SN) of

the central nervous system of humans and other phylo-

genetically close to mammals including the chimpanzee,

gibbon, and baboon and more distant ones, such as

horses and sheep (Marsden, 1961).

Neuromelanin is produced after the first 2–3 yr of life

(Cowen, 1986; Mann and Yates, 1974; Zecca et al., 2001)

and accumulates with aging (Bogerts, 1981; Graham,

1979; Mann and Yates, 1974; Sulzer et al., 2008; Zecca

et al., 2002, 2008b). NM differs from the peripheral

Significance

In contrast to the cutaneous melanin, the biosynthesis and structure of neuromelanin (NM) still remain

poorly understood. To mimic the processes of NM synthesis and its aging, we performed model

experiments starting with tyrosinase oxidation of various ratios of DA and Cys followed by heating the

synthetic NMs. Time course of the NM synthesis and its alteration by heating was followed by chemical

degradation methods. Natural NM was found to correspond to a melanin prepared from a 2:1 molar ratio of

DA and Cys followed by heating at 37°C for 40 days.

792 ª 2012 John Wiley & Sons A/S

Pigment Cell Melanoma Res. 25; 792–803 ORIGINAL ARTICLE

melanins as it predominates in catecholaminergic neu-

rons of the SN and the locus coeruleus and is formed by

the oxidation of catecholamines, dopamine (DA), and

noradrenaline. These areas in the human brain undergo

severe degeneration during the progression of

Parkinson’s disease (PD) (Zarow et al., 2003; Zecca et al.,

2002). However, the biosynthesis, structure, and function

of NM are not well characterized in spite of the possible

involvement of NM in the etiology and pathogenesis of

PD (Smythies, 1996). Histochemical studies on human

SN and locus coeruleus reported that NM has character-

istics similar to peripheral melanins (Barden, 1975; Van

Woert and Ambani, 1974).

Several hypotheses have been proposed for the roles

of NM: it may play a cytoprotective role by sequestering

redox-active metals (Fe, Cu, Mn, and Cr), toxic metals

(Cd, Hg, and Pb) (Zecca et al., 1994, 1996, 2008b),

organic toxic compounds such as MPP+ (D’Amato et al.,

1986), and pesticides with an environmental risk factor for

PD (Lindquist et al., 1988). NM also provides a protective

mechanism against DA toxicity, preventing the cytosolic

accumulation of cytotoxic DAquinone produced by DA

oxidation (Sulzer et al., 2000). Zareba et al. (1995) and

Zecca et al. (2008a) showed that the blockade of iron into

a stable iron–NM complex inhibits the formation of

neurotoxic DAquinone. These processes occur intraneur-

onally during aging and in PD, thus showing that NM is

neuroprotective. NM might also become a source of free

radicals by reaction with hydrogen peroxide (H2O2) (Zecca

et al., 2008a). When the load of Fe3+ increases in NM, the

production of toxic-free radicals is catalyzed as shown in

studies using synthetic DA melanin (Ben-Shachar et al.,

1991; Zareba et al., 1995) and in studies using human NM

(Fasano et al., 2006; Zecca et al., 2008a). It has been also

reported that synthetic DA melanin increases the vulner-

ability of SN neurons (Offen et al., 1997).

Previous studies from our group and other groups have

shown that NM consists of complex polymers derived

from eumelanic DHI and pheomelanic benzothiazine units

along with additional unidentified structural units (Aime

et al., 1994; Double et al., 2000; d’Ischia and Prota, 1997;

Wakamatsu et al., 1991, 2003). Dzierzega-Lecznar et al.

(2006) demonstrated that pyrolysis–gas chromatography/

mass spectrometry is suitable for identification and

differentiation of eumelanin derived from DA and pheo-

melanin derived from cysteinyldopamine (CDA). A Swed-

ish group (Rosengren et al., 1985) first detected 5-S-

cysteinyldopamine (5-S-CDA) in the human SN, suggest-

ing the possible incorporation of 5-S-CDA into NM. These

authors then proposed that NM consists of about equal

amounts of CDA-derived and DA-derived units, based on

the results of chemical degradation studies (Carstam

et al., 1991; Odh et al., 1994). Subsequently, we inves-

tigated the structure of NM using alkaline H2O2 oxidation

and reductive hydroiodic acid (HI) hydrolysis to evaluate

the composition of human NM (Wakamatsu et al., 2003).

In the present study as well as in the previous one, we

used the alkaline H2O2 oxidation method to quantitate

DA-derived DHI units (Ito and Wakamatsu, 1998). That

method was recently improved to make it more simple

and reproducible (Ito et al., 2011). Alkaline H2O2 oxidation

of DA melanin yields pyrrole-2,3-dicarboxylic acid (PDCA)

and pyrrole-2,3,5-tricarboxylic acid (PTCA) as degradation

products of DHI-derived units depending on the absence

or presence of a connection at the C2 position,

respectively (Figure 1). In addition, CDA melanin yields

thiazole-4,5-dicarboxylic acid (TDCA) and thiazole-2,4,5-

tricarboxylic acid (TTCA) as degradation products of

Figure 1. Chemical degradation products

from DA-derived melanin and from CDA-

derived melanin.

ª 2012 John Wiley & Sons A/S 793

Biosynthetic pathway and aging process of neuromelanin

benzothiazole units. On HI hydrolysis, CDA melanin

affords 4-amino-3-hydroxyphenylethylamine (4-AHPEA)

and 3-amino-4-hydroxyphenylethylamine (3-AHPEA) from

benzothiazine-derived units (Wakamatsu et al., 2003),

while 6-(2-aminoethyl)-4-hydroxy-benzothiazole (BZ-1) is

formed from benzothiazole-derived units. Based on the

results of those chemical degradations, we previously

suggested that NM is derived mostly from DA with 21 to

25% incorporation of CDA-derived units into NM (Wak-

amatsu et al., 2003). However, in that study, melanin

contents in NM were found to be 11–13% based on

spectrophotometric methods while they were only 1.7%

based on chemical degradation methods. Furthermore,

there was a large difference in 4-AHPEA values between

natural NM and synthetic NM. In addition to the melanic

component, human NM contains lipids of polyisoprenic

type and a peptide component (Fedorow et al., 2006;

Zecca et al., 2000, 2008b).

To elucidate the physiological roles of NM during the

aging process and its pathophysiological roles in the

etiology of PD, it is essential to understand more about

the structure and biosynthesis of NM and its degradation

in vivo. Pheomelanic units in NM are derived from CDA,

mostly 5-S-CDA (Figure 2). The 7-(2-Aminoethyl)-5-

hydroxy-3,4-dihydro-2H-1,4-benzothiazine- 3-carboxylic

acid (DHBT-1) has been suggested as an intermediate

following 5-S-CDA (Li and Dryhurst, 1997; Shen et al.,

1997). In analogy to the late stage of pheomelanin

production (Wakamatsu et al., 2009), we may be able to

identify 3-oxo-3,4-dihydro-1,4-benzothiazine (ODHBT-1),

secondary modification of the benzothiazine moiety (BT-

1), and BZ-1 in the course of the biosynthesis of NM. We

also hypothesize that the discrepancies raised in our

previous studies might be derived from the aging of

natural NM in vivo. To test this hypothesis and to

elucidate the biosynthetic pathway of NM, we have

carried out the following two sets of experiments: (i)

elucidation of the biosynthetic pathway of NM more

precisely by analyzing the time course of oxidation of DA

in the presence of various ratios of Cys and (ii) elucidation

of structural alterations of NM during the aging process

by analyzing the time course of changes in degradation

markers from heated synthetic NM.

Results

Preparation of putative NM precursors

To follow the biosynthetic pathway for the pheomelanic

moiety of NM, we synthesized putative precursors

(intermediates) (Figure 2). In addition to previously known

5-S-CDA and DHBT-1, we were able to newly prepare

ODHBT-1 and BZ-1 by following methods reported

previously for the corresponding amino acid derivatives

(Wakamatsu et al., 2009).

Analysis of natural NM

Chemical degradation analyses of natural NM using

alkaline H2O2 oxidation and HI reductive hydrolysis were

performed (Figure 1). Following alkaline H2O2 oxidation,

TDCA and TTCA were produced at 3.35 and 5.56 nmol/

mg as averages for two samples, respectively (Table 1).

PDCA and PTCA were minor products (1.18, and

0.29 nmol/mg, respectively). Following HI reductive

hydrolysis, 4-AHPEA and 3-AHPEA were found at 3.44

Figure 2. Biosynthetic pathway of the

pheomelanic moiety of NM. Only products

derived from 5-S-CDA among its isomers

are shown. Products in parentheses are

those with a short half-life and are not

isolable.

794 ª 2012 John Wiley & Sons A/S

Wakamatsu et al.

and 4.94 nmol/mg, respectively. The preferential produc-

tion of 3-AHPEA was confirmed as in our previous study

(Wakamatsu et al., 2003). Most 3-AHPEA seems to come

from 3-nitrotyramine. Tyramine is present in various

regions of the brain (Jones et al., 1983; Silkaitis and

Mosnaim, 1976), and nitration of tyramine is known to

proceed in vivo (Rankin et al., 2008). BZ-1 was newly

detected in the present study at a level (4.99 nmol/mg)

similar to 4-AHPEA and 3-AHPEA.

Elucidation of the biosynthetic pathway of NM

Tyrosinase was employed as an oxidant in this study, as it

gradually and constantly produces DAquinone from DA

and the oxidation can easily been controlled. When

tyrosinase oxidation was performed with mixtures of

DA (1.00 mM) and Cys (1.00, 0.50 and 0.25 mM), the

oxidation mixtures turned to reddish brown between 1

and 15 min, depending on the molar ratio of DA to Cys.

Black melanin pigments began to deposit at 7–30 min,

and the supernatants turned almost colorless at 60 min.

The oxidation was followed by UV-VIS spectrophotometry

and HPLC analysis of the precursor DA and the products

CDA isomers and others. We did not to attempt to

separate insoluble pigment from soluble precursor and

intermediates because the whole process proceeded

rapidly and continuously. Figure 3 summarizes the time

course of the tyrosinase oxidation of mixtures from 1:1,

1:0.5, and 1:0.25 molar ratios of DA and Cys. As shown in

Figure 3A, during the oxidation of a mixture of 1:1 ratio of

DA and Cys, DA was gradually consumed over 0–15 min,

during which time CDA isomers accumulated (ca. 40%)

and then decreased at 7 min. Concomitant with the

disappearance of CDA, a new compound DHBT-1 began

to appear at 3 min, reached a maximum at 7 min (ca.

30%), and then disappeared at 15 min. Thus, a soluble

pheomelanic pigment seems to have appeared at this

first stage. After exhaustion of Cys, the remaining DA

was oxidized and disappeared within 15 min with a

gradual increase in visible absorption, and an insoluble

eumelanic pigment began to precipitate around 20 min.

BZ-1 and ODHBT-1 were detected in low yields at the

later stage of oxidation (BZ-1 in ca. 2% at 60 min). The

absorbance at 500 nm increased along with the deposi-

tion of melanin pigment. During the oxidation of a mixture

of 1:0.5 molar ratio of DA and Cys (Figure 3B), CDA

appeared at 1 min (ca. 20%) and then decreased. DHBT-1

was detected between 1 and 7 min. BZ-1 was detected

throughout the oxidation in trace yields (<0.5%). The

absorbance at 500 nm increased from the beginning of

the oxidation. During the oxidation of a mixture of 1:0.25

molar ratio of DA and Cys (Figure 3C), CDA and DHBT-1

were not detected even at 1 min owing to the rapid

oxidation process, while BZ-1 was detected and reached

a maximal yield (ca. 1%) at 7 min and thereafter

decreased. An insoluble eumelanic pigment appeared

around 10 min.

The time course of the oxidation was then followed by

alkaline H2O2 oxidation giving TTCA, TDCA, PTCA, and

PDCA. TTCA and TDCA (benzothiazole degradation prod-

ucts) showed gradual increases with 2.3 and 2.1% yields

from DA+Cys (1:1), 1.5 and 1.3% yields from DA+Cys(1:0.5), and 1.2 and 0.7% from DA+Cys (1:0.25) melanin,

respectively, at 60 min (Figure 4A–C). PTCA and PDCA

showed rather constant yields throughout the oxidation.

Reductive hydrolysis with HI was performed to follow the

production of 4-AHPEA and 3-AHPEA. AHPEA isomers

were produced from the beginning of the reaction and

gradually decreased to a half at 60 min except for DA:Cys

Table 1. Chemical characterization of natural NM in comparison with model melanins

Method Analyte

NM 1 NM 2

DA + Cys

(1:0.5)-melanina

Heated

DA + Cys

(1:0.5)-

melaninb

(nmol/mg) (nmol/mg)c

Soluene-350solubilization A500 9 1000 760 1080 6050 6743

HI hydrolysis 4-AHPEA 2.59 4.28 577 99.3

3-AHPEA 3.66 6.21 166 63.8

BZ-1 4.12 5.86 177 95.6

H2O2 oxidation TDCA 2.60 4.10 59.1 116

TTCA 4.34 6.77 62.8 140

PDCA 0.90 1.45 33.9 31.0

PTCA 0.28 0.30 9.76 9.99

Ratio TTCA/4-AHPEA 1.68 1.58 0.11 1.41

4-AHPEA/PDCA 2.88 2.95 17.0 3.20

TTCA/PDCA 4.82 4.67 1.85 4.50

aValues are before heating at 37°C.bValues are after heating at 37°C for 40 days.cValues were calculated, assuming that the amount of DA+Cys (1:0.5)-melanin obtained from 0.01 mmol DA were 2.13 mg (based on the

molecular weights of DA and CDA).

ª 2012 John Wiley & Sons A/S 795

Biosynthetic pathway and aging process of neuromelanin

(1:1) melanin which showed an increase up to 7 min

(Figure 4D–F). 3-AHPEA was produced as a minor

isomer, and the ratio of 4-AHPEA to 3-AHPEA decreased

a little from 5.3 to 4.4 (DA:Cys = 1:1) during 60 min of the

oxidation. The benzothiazole-intermediate BZ-1 was also

produced at 7-15 min and reached maximal yields of 5.9,

1.8, and 1.3% from DA+Cys (1:1), DA+Cys (1:0.5), and

DA+Cys (1:0.25) melanin, respectively, at 60 min (Fig-

ure 4D–F). These results clearly indicate that benzothi-

azine units (AHPEA) are rather rapidly converted to

benzothiazole units (TDCA, TTCA, and BZ-1) during the

time course of 60 min.

Elucidation of the aging process of synthetic NM

During the biosynthetic study of melanic component of

NM, we observed time-dependent changes of their

structural features, especially the conversion from

benzothiazine to benzothiazole units. This ‘aging’ process

was examined in more detail to mimic the aging process

of natural NM that may occur in the human brain. Aging

simulation of synthetic NM prepared from DA+Cys (1:1,

1:0.5, and 1:0.25) were performed under heating of the

oxidation mixtures (suspensions) at three different con-

ditions: 37°C for up to 120 days, 100°C for up to 24 h, and

60°C for up to 24 days. Figure 5 summarizes the results

of heating at 37°C of melanin prepared from DA+Cys(1:1), DA+Cys (1:0.5), and DA+Cys (1:0.25). Both TTCA

and TDCA showed gradual increases, but TTCA increased

at greater rates (Figure 5A–C). PTCA and PDCA showed

only slight decreases. As shown in Figure 5D–F, 4-

AHPEA and 3-AHPEA rapidly decreased, and their ratios

also decreased gradually from 3.7 to 2.6. BZ-1 also

decreased gradually. Degradation products from DA+Cys-melanins heated at 100°C for up to 24 h also showed

A B

C

Figure 3. Time course of production of

melanogenic intermediates of NM from DA

in the presence of Cys. (A) DA+Cys (1:1),

(B) DA+Cys (1:0.5), (C) DA+Cys (1:0.25).

Consumption of DA, production/

consumption of CDA and DHBT-1, and

production of BZ-1 and ODHBT-1 were

analyzed. Pheomelanin is formed first from

DA and Cys, and after the depletion of Cys,

eumelanin from DA begins to deposit on

the preformed pheomelanin to give an

insoluble pigment. Percent values were

calculated on a molar basis against DA

(1.0 mM). Total melanin (A500) was

calculated by multiplying absorbances at

500 nm by 20. The reaction was performed

on two separate occasions with similar

results.

A B C

D E F

Figure 4. Time course of production of DA

+Cys-melanin. Alkaline H2O2 oxidation of

(A) DA+Cys (1:1)-melanin, (B) DA+Cys(1:0.5)-melanin, (C) DA+Cys (1:0.25)-

melanin. TDCA, TTCA, PDCA, and PTCA

were analyzed. HI hydrolysis of (D) DA+Cys(1:1)-melanin, (E) DA+Cys (1:0.5)-melanin,

(F) DA+Cys (1:0.25)-melanin. 4-AHPEA, 3-

AHPEA, and BZ-1 were analyzed. Percent

values were calculated on a molar basis

against DA (1.0 mM). The reaction was

performed on two separate occasions with

similar results.

796 ª 2012 John Wiley & Sons A/S

Wakamatsu et al.

similar patterns of continuous change (Figure S1). Heating

at 60°C for up to 24 days also gave results similar to

Figure 5 and Figure S1 (data not shown).

As 4-AHPEA and TTCA are the major degradation

products deriving from pheomelanin and showed dra-

matic changes in yield during aging, we examined the

time course of changes in the TTCA/4-AHPEA ratio from

DA+Cys-melanins heated at 37°C. The ratios of TTCA to

4-AHPEA increased about 10-fold in proportion to the

heating time irrespective of the ratio of DA to Cys

(Figure 6A). This result clearly indicates that benzothi-

azine units are gradually converted to benzothiazole units

during the aging process even at 37°C in a time span of

up to 120 days. Thus, the TTCA/4-AHPEA ratio serves as

a good indicator of the aging process of the pheomelanic

moiety of NM.

We next tried to compare the structural features of

natural NM to aged synthetic NM. For this purpose, we

examined changes in yields of the major degradation

products, 4-AHPEA and TTCA, in relation to yields of

PDCA as changes in PDCA yield were minimal. Figure 6B

shows the ratios of three markers during the aging

process of synthetic NM at 37°C. The ratio of 4-AHPEA to

PDCA represents the relative ratio of the benzothiazine

moiety in NM, while the ratio of TTCA to PDCA

represents the relative ratio of the benzothiazole moiety

in NM. By heating NM at 37°C for 120 days, the 4-

AHPEA/PDCA ratio decreased continuously while the

TTCA/PDCA ratio reached a maximum at 40 days and

thereafter decreased a little, irrespective of the ratio of

DA to Cys. When we compared these with the ratios

from natural NM, these results indicate that the structure

of melanic component of natural NM is close to those

prepared by heating synthetic NM from DA+Cys (1:0.5) at

37°C for 40 days (Figure 6B), at 100°C for 8 h (Figure

S1B), or at 60°C for 8 days (data not shown).

Table 1 summarizes the chemical characterization of

human NM in comparison with synthetic DA+Cys (1:0.5)

melanin before and after heating at 37°C for 40 days. The

isolated human NM gives 4-AHPEA/TTCA, 4-AHPEA/

PDCA, and TTCA/PDCA ratios close to those of heated

synthetic NM but not to non-heated synthetic NM (of any

ratio of DA to Cys). By comparing the absorbance at

500 nm, isolated NM contains 11.9% melanin, while the

D E

A B

F

C

Figure 5. Time course of the aging

process at 37°C for synthetic NM prepared

from various ratios of DA+Cys. AlkalineH2O2 oxidation of (A) DA+Cys (1:1)-

melanin, (B) DA+Cys (1:0.5)-melanin, and

(C) DA+Cys (1:0.25)-melanin. TDCA, TTCA,

PDCA, and PTCA were analyzed. HI

hydrolysis of (D) DA+Cys (1:1)-melanin, (E)

DA+Cys (1:0.5)-melanin, and (F) DA+Cys(1:0.25)-melanin. 4-AHPEA, 3-AHPEA, and

BZ-1 were analyzed. Percent values were

calculated on a molar basis against DA

(1.0 mM). The chemical degradation was

performed in duplicate.

A B

Figure 6. Time course of changes in the

ratios of melanin degradation products

during the aging process of synthetic NM

prepared from various ratios of DA+Cys at

37°C. (A) TTCA/4-AHPEA ratio. (B) 4-

AHPEA/PDCA and TTCA/PDCA ratios. The

two yellow circles represent values from

natural NM.

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Biosynthetic pathway and aging process of neuromelanin

percent contents are 3.1, 3.3, and 3.5% based on the

values of 4-AHPEA, TTCA, and PDCA, respectively.

Discussion

NM predominates in catecholaminergic neurons of the

SN and the locus coeruleus and differs from peripheral

melanins produced in the hair, skin, and eyes (Bogerts,

1981; Double et al., 2011; Saper and Petito, 1982; Zecca

et al., 2003). It was suggested that NM is formed by the

oxidation of catecholamines (Bogerts, 1981; Fedorow

et al., 2005; Graham, 1979; Mann and Yates, 1974;

Napolitano et al., 2011). Studies on cultures of dopami-

nergic neurons have demonstrated that NM is generated

by oxidation of cytosolic DA (Sulzer et al., 2000).

Although low levels of tyrosinase mRNA have been

found in human SN (Xu et al., 1997), tyrosinase protein

does not appear to be expressed in this brain region

(Ikemoto et al., 1998). Thus, current evidence suggests

that tyrosinase does not play a role in the synthesis of

NM in the human brain. Other enzymatic pathways have

been proposed for NM synthesis, such as tyrosine

hydroxylase (Haavik, 1997), peroxidase (Okun, 1997),

prostaglandin H synthase (Hastings, 1995; Mattammal

et al., 1995), and macrophage migration inhibitory factor

(Matsunaga et al., 1999). NM could alternatively be

derived from the non-enzymatic autooxidation of cate-

cholamines to ortho-quinones with addition of a thiol

group, as this reaction has been demonstrated in the

brain (Fornstedt et al., 1986). Thus, the NM biosynthetic

pathway is diverted by scavenging DAquinone (Zhang

and Dryhurst, 1993) which undergoes nucleophilic attack

by the thiol group of L-Cys/GSH to give 5-S-CDA/5-S-

glutathionyldopamine, the latter being subsequently

hydrolyzed by peptidase enzymes to 5-S-CDA (Napolitano

et al., 2011). In synaptic vesicles, DA is normally taken up

by the vesicular monoamine transporter VMAT2, and

overexpression of VMAT2 is protective against DA

derived from toxic compounds (Munoz et al., 2012;

Sulzer et al., 2000). Excess DA can interact with Fe3+

to form quinones and semiquinones (Zecca et al., 2008a).

These reactive intermediates are known to generate free

radicals and induce lesions of nerve cells (Arriagada et al.,

2004; Sulzer and Zecca, 2000). If these reactive interme-

diates are trapped by Cys to form CDA with subsequent

conversion to NM, neurodegeneration could be avoided.

Basic information on the biodegradative pathway of

neuromelanin is lacking. It is therefore essential to know

what kind of structural alterations would occur during

thermal degradation of neuromelanin mimicking aging

process. However, it is well known that the presence of

iron accelerates autoxidation of DA, leading to the

formation of neurotoxins like tetrahydroisoquinolines

(Naoi et al., 1995; Napolitano et al., 1999, 2011). There-

fore, it would be interesting to evaluate the effects of iron

ions or other metal ions and exposure to reactive oxygen

species on the course of biodegradative pathway in

comparison with the results obtained in this chemical

study. These will be left for our future study.

NM synthesis is a neuroprotective process. In fact, it

was shown that NM synthesized in various regions of the

human brain plays an important role in the protective

process because the melanic component is generated

through the removal of reactive/toxic quinones that

otherwise would cause neurotoxicity (Sulzer et al.,

2000; Zecca et al., 2008b). Those toxic compounds

include DAquinone, CDA, and DHBT-1 (Arriagada et al.,

2004; Graham et al., 1978; Li and Dryhurst, 1997; Shen

and Dryhurst, 2001; Spencer et al., 2002). DAquinone

was found to cyclize ca. 100-fold more slowly to form an

aminochrome than does dopaquinone (ortho-quinone of

3,4-dihydroxyphenylalanine), rendering it highly reactive

and thus toxic (Ito and Wakamatsu, 2008; Land and Riley,

2000; Segura-Aguilar et al., 2001). Thus, DA may exert its

toxicity through oxidation to DAquinone followed by

binding to SH enzymes essential for cell proliferation

and survival. CDA is a product of detoxification of

DAquinone, but itself is neurotoxic (Spencer et al.,

2002). In fact, levels of CDA are elevated in the brains

of patients who died from PD (Vauzour et al., 2008). 5-S-

CDA is more readily oxidized than its parent catechol-

amine precursor DA and is converted to DHBT-1 that is

also toxic to cells of the SN (Vauzour et al., 2008, 2010).

Thus, the sequence of reactions starting with DA oxida-

tion, the production of DAquinone, CDA and DHBT-1, is

likely a two-edged sword of toxic and detoxifying

processes. The toxicity results from the facile autoxida-

tion of these intermediates, thus producing reactive

oxygen species (ROS). However, cells in the SN would

be protected by incorporating these toxic intermediates

into ‘insoluble’ NM. The present study has clearly shown

that CDA and DHBT-1 are incorporated (and disappear)

into growing ‘insoluble’ NM. In this regard, there is a

hypothesis that NM has a cytoprotective function in the

sequestration of redox-active metal ions under normal

conditions but it has a cytotoxic role in the pathogenesis

of PD (Enochs et al., 1994).

Our present study indicates that NM in the SN may be

produced from DA and Cys in a molar ratio of about 2:1,

and the continuous alteration in the structure of NM

proceeds rather extensively during the aging process in

which most benzothiazine moieties are converted to

benzothiazole moieties. This study also provides evi-

dence for the biosynthetic pathway that the CDA-derived

pheomelanic moiety of NM is produced in the sequence

of 5-S-CDA, DHBT-1, ODHBT-1, and/or BZ-1 (Figure 3).

Biosynthetic and biodegradative pathways are a con-

tinuous process, but in this study we tried to differentiate

the two pathways by making the former pathway much

faster than the latter with the use of tyrosinase as a

chemical-oxidizing agent. Autooxidation of DA (in the

presence of Cys) proceeds much slower than the oxida-

tion with tyrosinase (Greco et al., 2011), leading to

ambiguous situations regarding the ratio of DA and Cys

798 ª 2012 John Wiley & Sons A/S

Wakamatsu et al.

incorporated into insoluble pigments (owing to oxidation

to cystine) and the differentiation between biosynthetic

and biodegradative pathways. CDA formation also occurs

under non-enzymatic conditions. The pathway of oxida-

tion of catecholamine using tyrosinase is known to be

similar to ones under non-enzymatic (Napolitano et al.,

2011; Palumbo et al., 1995) or other enzymatic conditions

(Haavik, 1997; Hastings, 1995; Matsunaga et al., 1999;

Mattammal et al., 1995; Okun, 1997).

In our previous study (Wakamatsu et al., 2003), we

noticed some discrepancies in the structural features of

synthetic and natural NM. One is the large difference in

melanin contents of natural NM based on spectrophoto-

metric methods (11–13%) compared with chemical deg-

radation methods (1.7%). Based on the assumption that

this discrepancy might be derived from changes in the

structural features of natural NM in vivo, we performed

experiments to simulate aging by heating synthetic NM

obtained from various ratios of DA+Cys. In this experi-

ment, 4-AHPEA was observed to decrease rapidly with a

gradual increase of TTCA (Figure 4). This is the first study

showing that the benzothiazine moiety in synthetic NM is

gradually converted to the benzothiazole moiety by

heating that mimics the aging process of natural NM.

Thus, the ratio of TTCA to 4-AHPEA can be used as a

marker for the aging process of natural NM (Figure 6,

Figure S2). The present study shows that natural NM is

close in structural features to synthetic NM prepared

from a 2:1 mixture of DA to Cys and heated for 40 days at

37°C, for 8 h at 100°C (Figure 6, Figure S2), or for 8 days

at 60°C (data not shown). The melanin content in this

study was 11.9% based on the spectrophotometric

method while it was 3.1–3.5% based on chemical

degradation methods (Table 1). There still remains some

discrepancy between those two methods. The threefold

higher content by spectrophotometry suggests that there

might be unknown pigmented materials other than the

(aged) melanin moiety in natural NM. The reason for

these discrepancies could be that in natural NM, in

addition to ‘normal’ melanin, another oligomeric structure

is present, something similar to melanin but lacking some

of the typical characteristics associated with known

melanic structures. Another possible explanation for the

higher content by spectrophotometry may be that some

kinds of lipid components including the isoprenoid

dolichol (comprising 35% of NM) or proteinaceous com-

ponents of NM like a-synuclein darken by conjugation

with melanin in aging NM (Fasano et al., 2003; Fedorow

et al., 2006). These possibilities will be addressed in our

future studies.

Regarding the three-dimensional structure of NM, Bush

et al. (2006) showed that human NM is composed of

granules whose size is 200–500 nm, and these contain

subunits of ~30 nm diameters consisting of pheomelanin

at the core and eumelanin at the surface (Ito, 2006). The

present study has given further evidence in support of

this ‘casing’ model (Ito and Wakamatsu, 2008). DA was

firstly consumed followed by the accumulation and

decrease of CDA. After exhaustion of Cys, the remaining

DA was oxidized, and an insoluble eumelanic pigment

precipitated. This shows that the soluble pheomelanin

core is first produced at the beginning of NM production,

which is followed by the production and deposition of

insoluble eumelanic pigment on the core (Figure 3),

concomitant with the gradual conversion of benzothiazine

to the benzothiazole moiety (Figure 4). It is interesting to

speculate that a peroxidative insult on NM granules in

neurons of the SN would degrade the eumelanin surface,

thus exposing the ‘soluble’ pheomelanic core. If this

event would happen, NM granules would be solubilized to

release soluble pheomelanic pigment, known to be

cytotoxic (Simon and Peles, 2010).

In conclusion, we demonstrate the instability of the

benzothiazine unit in NM during the aging process. We

have published that a similar process of conversion of

benzothiazine to benzothiazole proceeds in pheomelanin

of human red hairs under heating conditions (Ito et al.,

2011) as well as under UVA irradiation (Wakamatsu et al.,

2012). It is likely that the conversion of benzothiazine to

benzothiazole moiety of NM may render NM more stable

and less toxic. In the past, most in vitro studies employed

DA melanin as a model NM (Ben-Shachar et al., 1991;

Offen et al., 1999), despite the considerable disparity

between natural and synthetic NM (Zecca et al., 2008a).

A method to prepare synthetic NM that structurally

mimics the melanic component of human NM is now

established, that is, tyrosinase oxidation of DA+Cys in a

ratio of 2:1 followed by heating at 37°C for 40 days (for

the sake of convenience, at 100°C for 8 h). This prepa-

ration of model NM should facilitate studies elucidating

the physiological roles of NM during the aging process

and its role in the pathogenesis of PD.

Methods

All chemicals were of the highest purity available. DA, Cys, mush-

room tyrosinase (EC.1.14.18.1, 5370 units/mg), and 57% HI were

purchased from Sigma-Aldrich (St Louis, MO, USA). Fifty per cent

H3PO2, 30% H2O2, and sodium 1-octanesulfonate were purchased

from Nacalai Tesque, Inc (Kyoto, Japan). Soluene-350 was purchased

from Perkin-Elmer (Waltham, MA, USA). Preparations of PTCA,

PDCA, TTCA, TDCA, 4-AHPEA, and 3-AHPEA were carried out as

described previously (Ito and Wakamatsu, 1998, 2003; Wakamatsu

et al., 2003). 5-S-CDA was prepared as described by Ito et al. (1986).

UV-visible spectra were recorded with a JASCO V-520 UV/VIS

spectrophotometer (Tokyo, Japan). Mass spectra were analyzed

using electrospray ionization ion trap mass spectrometry (ESI-ion trap

MS) (LCQ DECA XP, Thermo Fisher Scientific K.K., Yokohama, Japan)

at the Institute for Comprehensive Medical Science of our University.

Two natural NMs from human SN were prepared as described

previously (Zecca et al., 2008b). Each of them was prepared from a

pool of 5 human SN (average age: 82 and 75 yr old, respectively). This

study was approved by the ethical committee of the National

Research Council of Italy–Institute of Biomedical Technologies

(Segrate, Milan, Italy) and was carried out in agreement with the

Policy of National Research Council of Italy.

ª 2012 John Wiley & Sons A/S 799

Biosynthetic pathway and aging process of neuromelanin

HPLC conditions

Determination of melanin markers was performed using HPLC as

previously reported (Ito and Wakamatsu, 1998; Ito et al., 2011;

Wakamatsu et al., 2002, 2003). For analysis of DA, 5-S-CDA, 4-

AHPEA, and 3-AHPEA by HPLC, we used methods previously

described with minor modifications of HPLC conditions: the mobile

phase was 0.1 M sodium citrate buffer, pH 3.0, containing 1 mM

sodium octanesulfonate and 0.1 mM EDTA.2Na: methanol, 90:10 (v/

v) (Wakamatsu et al., 2003). Analyses were performed at 40°C.Analysis of TDCA, TTCA, PDCA, and PTCA by HPLC was performed

as described in Ito et al. (2011). For the analysis of DHBT-1, ODHBT-

1, and BZ-1, we used a Capcell pak C18 column with 0.1 M

potassium phosphate buffer, pH 2.1: methanol, 92:8 (v/v) at 40°C,with a UV-VIS detector set at 254 nm for the determination of DHBT-

1 and ODHBT-1 and with an electrochemical detector set

at + 900 mV versus Ag/AgCl electrode for BZ-1, at a flow rate of

0.7 ml/min. The retention times of DHBT-1, ODHBT-1, and BZ-1

were 29.4, 23.9, and 17.7 min, respectively. For the preparative

separation of BZ-1, we used a Capcell pak C18 column (type MG,

20 9 250 mm, 5 lm particle size, from Shiseido), with 0.1 M

potassium phosphate buffer, pH 2.1: methanol, 85:15 (v/v), at 55°C, with a UV-VIS detector set at 256 nm, at a flow rate of 5.0 ml/min.

Synthesis of DHBT-1

DHBT-1 was prepared by a method (Wakamatsu et al., 2009) similar

to that reported for the dihydrobenzothiazine derivative of 5-S-CDA

(Li and Dryhurst, 1997). A solution of 5-S-CDA (0.10 mmol) in 10 ml

0.05 M sodium phosphate buffer, pH 6.8, was raised to pH 9.0 with

2 M NaOH. The solution was stirred for 10 min at 25°C and was then

acidified to pH 3 with 2 M HCl. The mixture was passed through a

column (1.0 9 5.0 cm in water) of Dowex 50W-X2, and DHBT-1 was

eluted with 3 M HCl after washing with 0.5 M HCl. Fractions

containing DHBT-1 were evaporated to dryness in vacuo and then

subjected again to Dowex 50W-X2 chromatography (1.0 9 10.5 cm

in 3 M HCl). The 2HCl salt of DHBT-1 was obtained as an almost

colorless glassy powder, which was further purified by recrystalliza-

tion from ethanol–ether to give 24.0 mg (69% yield). UV kmax 294 (e1520) and 232 nm (11 780) in 0.1 M HCl. Mass spectrum: m/z 255

(M+H)+, 238, 209, 196, 192.

Synthesis of ODHBT-1

ODHBT-1 was prepared following the method for ODBT amino acid

(Wakamatsu et al., 2009). A reaction mixture of 5-S-CDA

(0.20 mmol) in 10 ml 0.1 M NaOH was vigorously stirred for

60 min at 25°C. The dark brown solution was acidified to pH 3.0

with 400 ll 3 M HCl, and the mixture was passed through a column

(1.0 9 5.0 cm in water) of Dowex 50W-X2, and ODHBT-1 was

eluted with 3 M HCl after washing with 0.5 M HCl. Fractions

containing ODHBT-1 were evaporated to dryness in vacuo and then

subjected again to Dowex 50W-X2 chromatography (1.0 9 6.3 cm in

3 M HCl). The crystalline HCl salt of ODHBT-1 was further purified by

recrystallization from ethanol—ether to give 13.0 mg (19% yield). UV

kmax 296 (e 4760) and 240 nm (16 400). Mass spectrum: m/z 225 (M

+H)+, 207, 197, 167.

Synthesis of BZ-1

BZ-1 was prepared from the HI hydrolysate of 5-S-CDA melanin

prepared according to the method of Wakamatsu et al. (2009) with

minor modifications. In brief, a mixture of CDA melanin (154 mg),

57% HI (10 ml), and 30% H3PO2 (1 ml) was heated under reflux for

20 h. HI was removed with a rotary evaporator, and the residue was

passed through a column (1.0 9 7.0 cm in water) of Dowex 50W-X2,

and a fraction containing 4-AHPEA and BZ-1 was eluted with 3 M

HCl. The fraction containing BZ-1 was again subjected to Dowex

50W-X2 chromatography (1.0 9 17 cm in 3 M HCl). BZ-1 was

purified under the preparative HPLC conditions described above.

Separated BZ-1 was desalted through Dowex 50W-X2 chromatogra-

phy (1.8 9 5.0 cm in 3 M HCl). Crystals of the HCl salts of BZ-1 were

purified by crystallization from ethanol–acetone to give 1.4 mg of BZ-

1 (0.9% yield). UV of BZ-1 kmax 304 (e 2900) and 230 nm (14 060).

Mass spectrum: m/z 195 (M+H)+, 177, 149.

Elucidation of the biosynthetic process of NM

To follow the time course of the biosynthetic pathway of NM, 0.1 mg

mushroom tyrosinase was added to a solution of DA (0.01 mmol) and

L-cysteine (0.01, 0.005, or 0.0025 mmol) in 10 ml 0.05 M sodium

phosphate buffer, pH 7.4, at 37°C. The oxidation was carried out

under the condition at 60 reciprocal rotations/min (with a 4 cm

stroke) with a regular shaker in a 100-ml Erlenmyer flask. Four

hundred microlitre of the reaction mixture was withdrawn at

predetermined times for the following assays: (i) for HPLC analysis

of DA, 5-S-CDA, DHBT-1, ODHBT-1, and BZ-1, 100 ll of the reaction

mixture was diluted 10-fold with 0.4 M HClO4, and the supernatant

was directly injected into the HPLC after centrifugation, (ii) alkaline

H2O2 oxidation and HI reductive hydrolysis were performed on a

100 ll aliquot of the reaction mixture using our reported methods (Ito

et al., 2011; Wakamatsu et al., 2002), (iii) absorbance at 500 nm

(total melanin) of a solution prepared from 900 ll Soluene-350 and a

100 ll aliquot of the reaction mixture (Ozeki et al., 1996).

Elucidation of the aging process of synthetic NM

A synthetic NM suspension prepared by tyrosinase oxidation of

various ratios of DA and Cys at pH 7.4 was heated in an electric oven

at 37°C for 120 days, at 60°C for 24 days, or at 100°C for 24 h.

Aliquots of the mixture were withdrawn at predetermined times for

the following assays as described previously: (i) alkaline H2O2

oxidation, (ii) HI reductive hydrolysis, and (iii) total melanin.

Acknowledgements

This work was supported by a Japan Society for the Promotion of

Science (JSPS) grant (No. 21500358, 24500450) given to KW and SI

and in part by the Italian Ministry of Education, University, and

Research (MIUR) – Research Projects of National Interest (PRIN)

Project 20085SYP79 and MIUR – Medical Research in Italy (MERIT)

Project RBNE08ZZN7. Authors also thank the Section of Legal

Medicine and Insurances, Department of Human Morphology

and Biomedical Sciences, University of Milano, for providing brain

tissues.

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Supporting information

Additional Supporting Information may be found in the

online version of this article:

Figure S1. Time course of the aging process at 100°Cfor synthetic NM prepared from various ratios of DA+Cys.

Alkaline H2O2 oxidation of DA+Cys (1:1)-melanin, (B) DA

+Cys (1:0.5)-melanin, and (C) DA+Cys (1:0.25)-melanin.

TDCA, TTCA, PDCA and PTCA were analyzed. HI hydro-

lysis of (D) DA+Cys (1:1)-melanin. (E) DA+Cys (1:0.5)-

melanin and (F) DA+Cys (1:0.25)-melanin. 4-AHPEA, 3-

AHPEA, and BZ-1 were analyzed. Percent values were

calculated on a molar basis against DA (1.0 mM). The

chemical degradation was performed in duplicate.

Figure S2. Time course of changes in the ratios of

melanin degradation products during the aging process of

synthetic NM prepared from various ratios of DA+Cys at

100°C. (A) TTCA/4-AHPEA ratio. (B) 4-AHPEA/PDCA and

TTCA/PDCA ratios. The two yellow circles represent

values from natural NM.

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Biosynthetic pathway and aging process of neuromelanin