Summary of ICMS Journal Club presentation on Friday, June 25, 2021.
Title: Recent Advances in Melanin Chemistry
Speaker: Prof. Dr. Kazumasa Wakamatsu, Emeritus Professor of Chemistry, Institute for Melanin Chemistry, Fujita Health University
On Friday, June 25, by Zoom, Professor Wakamatsu gave a review type of presentation to the Journal Club of the Institute for Comprehensive Medical Science (ICMS) of Fujita Health University about melanin. Melanin has been the primary research topic of Professor Wakamatsu’s >30-year career (see Prof. Wakamatu’s Science Portrait) and his review summarized both his own work and that of other research groups.
To the participants, to prepare for his presentation, he had recommended reading two articles of his:
1. Wakamatsu, K., Zippin, J.H., Ito, S. Chemical and biochemical control of skin pigmentation with special emphasis on mixed melanogenesis. Pigment Cell Melanoma Res., 2021. (https://onlinelibrary.wiley.com/doi/epdf/10.1111/pcmr.12970)
2. Wakamatsu, K., Murase, T., Zucca, F.A., Zecca, L., and Ito, S. Biosynthetic pathway to neuromelanin and its aging process. Pigment Cell Melanoma Res., 25, 792-803, 2012. (https://onlinelibrary.wiley.com/doi/epdf/10.1111/pcmr.12014)
There were 11 participants, and the atmosphere was very relaxed. Because of the biochemical topic, I had invited two biochemists who do not usually visit our Journal Club, namely Prof. Taei Matsui of Fujita Health University, and my good friend Dr. Axel Karger who lives in Germany and is specialized in virology. Other than them, and of course Emeritus Prof. Wakamatsu, we had two more scientists with a strong background in biochemical research, namely Emeritus Prof. Toshiharu Nagatsu and Emeritus Prof. Shosuke Ito. Luckily for me (and maybe for several other participants), it did not become the hardcore biochemical lecture that I had feared, but an entertaining introduction into the world of melanin which for the largest part was well-suited for the average biomedical researcher. I am grateful to Prof. Wakamatsu for his efforts to make this beautiful presentation. The discussion afterward was very enjoyable, with almost everyone asking questions which were—in harmony—answered by both Prof. Wakamatsu and his career-long friend and current co-worker Prof. Ito. Later, Dr. Ryuichi Nakajima of Nagoya University (formerly at FHU) told me that the relaxed atmosphere reminded him of the journal club events that he experienced while in Canada.
Contents of the presentation
What are melanins?
The word “melanin” can refer to a group of high molecular weight, mostly black and brown, pigments formed through the oxidation and polymerization of phenolic compounds. The word melanin can also be more restrictively used for the large biopolymer pigments whose synthesis involves the oxidation of tyrosine or dopamine (DA) (Fig. 1). The latter category was the topic of Prof. Wakamatsu’s presentation and in humans, like in many other vertebrates, include eumelanin, pheomelanin, and neuromelanin. Different from pheomelanin and neuromelanin, sulfur is not a major component of eumelanin.
Eumelanin and pheomelanin: the melanins that color us
Eumelanin (dark brown to black) and pheomelanin (yellow to reddish-brown) are synthesized from tyrosine and are best known for their coloring of skin, hair, and eyes. There they exist in different concentrations and ratios, causing the differences in colors between humans (shown for human hair in Fig. 2). A primary function of melanin in the skin is the absorbance of UV-light energy while converting it to heat. In the skin, melanin is made by melanocytes in organelles called melanosomes which can be transferred to keratinocytes to give them protection. Sunlight induces DNA damage in the keratinocytes, which then release melanocyte-stimulating hormone (MSH) that activates melanocyte through their melanocortin 1 receptors (MC1R) to produce eumelanin; this explains tanning (getting darker skin upon exposure to the sun). People, and also mice, with defects in M1CR tend to have light skin and red hair as they predominantly make pheomelanin. If not balanced by eumelanin, pheomelanin causes oxidative damage, and (orange-colored) M1CR-knockout mice were proven to be more susceptible to carcinogenesis than albino mice that lacked both eumelanin and pheomelanin. Albino people and animals tend to lack tyrosinase, an enzyme necessary for the oxidation of tyrosine so that it can enter the synthesis routes for eumelanin or pheomelanin (Fig. 1).
There appear to have been strong selective pressures per world region to match the level of skin pigmentation with the level of sun exposure. Too much pigment would prohibit the synthesis of Vitamin D, which predominantly takes place in the skin and is driven by UV-light, and too little pigment would create susceptibility to UV-light-induced cancer. Professor Wakamatsu explained several factors that could affect skin color, and one that I found particularly interesting is that the pH of the melanosomes in dark-skinned people is neutral whereas in light-skinned people it is acidic. He explained that an acidic pH suppresses the synthesis of eumelanin.
Professor Wakamatsu also gave an example how hair color could change with age, namely in Japanese women. Graying at old age is caused by a decrease in the number of melanocytes and a depletion of melanocyte stem cells in hair follicles. However, what Prof. Wakamatsu and co-workers specifically investigated was the darkening of hair from childhood to adulthood. They concluded that hair color darkening by aging is caused by an increase in total melanin (TM) relating to differently shaped melanosomes that are larger, and by an increase in the ratio of the DHI moieties relative to the DHICA moieties (for the structure of the individual moieties see Fig. 1) in eumelanin (Fig. 3).
Prof. Wakamatsu also briefly talked about how the moieties/components of eumelanin or pheomelanin (both large biopolymers exhibiting some variation in their organization) can be determined by chemical degradation protocols specific for analysis of each melanin type. However, my understanding of this is too little for trying to summarize that here. To nevertheless provide the readers here with the correct information, I have asked Prof. Wakamatsu to write this down himself, which he kindly did. For his summaries of his biochemical analysis systems, see the end of this blog post.
Eumelanin analysis from fossils
Because Prof. Wakamatsu together with Prof. Ito established unique protocols for determining the chemical composition of melanins, and they participated in several high-profile studies in which they analyzed melanin in fossils (e.g., in dinosaurs) that in some cases were hundreds of millions of years old. Especially interesting findings were obtained for Ichthyosaurs, which are extinct marine reptiles that looked a bit like modern toothed whales. Prof. Wakamatsu and Prof. Ito found that Ichthyosaurs possessed a bodily distribution of melanosomes and eumelanin that indicated a dark-back versus light-belly countershading (as in, for example, killer whales) (Fig. 4), and their co-authors found that Ichthyosaurs possessed a fat layer indicating that the animals were warm-blooded (Lindgren et al. 2018).
Neuromelanin: dark matter believed to protect neurons
Neuromelanin is only found in the brain, predominantly in neurons of the brain regions substantia nigra (SN) and locus coeruleus (LC), which therefore are colored dark (Fig. 5). The synthesis of neuromelanin is independent of tyrosinase and involves dopamine (Fig. 1) and/or other catecholic molecules. In neurons, neuromelanin accumulates in specialized autolysosomes and there forms complexes with lipids, proteins, and metal ions (Fig. 6). The function of neuromelanin is not well known, but it is thought to be neuroprotective by incorporating reactive dopaquinone and chelating toxic metal ions. The neuromelanin concentration increases with age and in elderly people, besides having a protective function, may also have a harmful effect. Compared to eumelanin and pheomelanin, the function, components, or synthesis of neuromelanin are not well understood. Prof. Wakamatsu and Prof. Ito made major contributions to the understanding of neuromelanin chemistry by developing chemical degradation protocols for analysis and by establishing protocols for the artificial biochemical synthesis of “model neuromelanin”. For a correct explanation of this, please see at the end of the blog post the paragraphs written by Prof. Wakamatsu himself. Except for the function of neuromelanin in aging, Prof. Wakamatsu and Prof. Ito are also very interested in its effects on Parkinson’s disease, as in that disease the neuromelanin-containing neurons of the substantia nigra are being lost.
Questions by the participants
The audience had a wide variety of questions. In most cases, there were no simple straightforward answers possible, but we had interesting discussions. Some of the questions that I remember are:
- Why are pools in junior high school and high schools in Japan not covered for shade, although it is not unusual that the swimming club students swim several hours every day.
- What is the relation between vitamin D and skin phenotype? (the answer was that higher UV-light penetration permits more efficient UV-dependent transformation of circulating precursors in the skin, which is an essential first step in vitamin D synthesis)
- Is there a relationship between melanin, Vitamin D, and susceptibility to COVID-19?
- Can neuromelanin accumulations in the brain be seen with techniques such as MRI? (the answer was a simple Yes)
- Can skin color be affected by trying to somehow change the pH of the melanosomes (e.g., through changes of the skin pH)?
- Can the increased accumulation of neuromelanin in neurons of the elderly be halted?
- What is more primitive, eumelanin or pheomelanin?
- Is it certain that having red hair increases the risk for Parkinson’s disease? (the answer was that this is not very well established)
- Is glutamic acid associated with accumulations of neuromelanin? (the answer was yes, but that the form of this association is yet unclear)
- Do albino mice (which lack tyrosinase) experience other phenotypes from not having eumelanin or pheomelanin besides being white? (the answer was that they also have poor vision and hearing)
Apart from the questions, it was also very interesting to hear Prof. Nagatsu speak about his past work in plants. Data from his group suggested that the formation of dopamine in banana is catalyzed by a phenol oxidase which is different from animal tyrosine hydroxylase (Nagatsu et al., Enzymologia, 1972). This is an example of how plants and animals have different biochemical pathways and how plants do not synthesize the types of melanin found in mammals.
Biochemical methods developed by Prof. Wakamatsu and Prof. Ito for the analysis of melanins
(these last paragraphs were kindly provided by Prof. Wakamatsu for this blog)
About eumelanin and pheomelanin
Most natural melanin pigments consist of both eumelanin (EM) and pheomelanin (PM) (a concept of “mixed melanogenesis”). To characterize melanin and melanogenesis, we developed a microanalytical method to analyze EM and PM (Ito and Fujita, Analytical Biochemistry, 1985) based on the chemical degradation of melanin pigments followed by the analysis of the degradation products using high-performance liquid chromatography (HPLC). We recently established a more convenient method for the simultaneous measurement of EM and PM (Wakamatsu et al., Pigment Cell Research, 2002; Ito et al., Pigment Cell & Melanoma Research, 2011; Ito and Wakamatsu, JEADV, 2011; Ito et al., International Journal of Molecular Sciences, 2020). This method is based on alkaline hydrogen peroxide (H2O2) oxidation to generate the specific markers, pyrrole-2,3,5-tricarboxylic acid (PTCA), pyrrole-2,3-dicarboxylic acid (PDCA), thiazole-2,4,5-tricarboxylic acid (TTCA) and thiazole-4,5-dicarboxylic acid (TDCA). PTCA is a specific biomarker of DHICA units or 2-substituted DHI units, whereas PDCA is a specific biomarker for DHI-derived units in EM, while TTCA and TDCA are specific biomarkers for BZ-derived moieties in PM. In addition to PTCA and PDCA, pyrrole-2,3,4,5-tetracarboxylic acid (PTeCA) and pyrrole-2,3,4-tricarboxylic acid (isoPTCA) have also been detected in fossil ink sacs (Glass et al., PNAS, 2012; d’Ischia et al., Pigment Cell & Melanoma Research, 2013), which suggests extra cross-linking of DHI units during aging of the EM polymer. PM is more readily analyzed than EM, as it is more soluble than EM. Most PM can be dissolved in alkaline media and consist of oligomers formed from sulfur-containing units, mostly benzothiazine (BT) and benzothiazole (BZ) moieties. Analysis of BT-derived moieties in PM was performed using hydroiodic acid, yielding 4-amino-3-hydroxyphenylalanine (4-AHP) and its isomer 3-amino-4-hydroxyphenylalanine (3-AHP) (Ito and Fujita 1985, https://doi.org/10.1016/0003-2697(85)90150-2; Wakamatsu et al. 2002, https://onlinelibrary.wiley.com/doi/abs/10.1034/j.1600-0749.2002.02017.x). A spectrophotometric method was also developed to measure the total melanin content estimated by absorbance at 500 nm (A500), which measures the combined amount of EM and PM. This method also provides a rough estimate of the ratio of EM to PM by analyzing the ratio of absorbance values at 650/500 nm. Ito et al. (2018, https://doi.org/10.1111/pcmr.12673) recently developed a novel method of melanin characterization based on acid hydrolysis of melanins followed by alkaline H2O2 oxidation of tissue samples. This method provides more simplified HPLC chromatograms compared with conventional H2O2 oxidation owing to the removal of proteins and low-molecular-weight compounds. It is useful not only to characterize melanins but also to confirm the presence of trace levels of EM and PM in various tissues and fossil samples (McNamara et al. 2018, https://www.nature.com/articles/s41467-018-05148-x).
About neuromelanin
Unlike ordinary melanins, a pigment called neuromelanin (NM) is deposited in the dopaminergic and norepinephrinergic neurons of the substantia nigra (SN) and the locus coeruleus (LC) in the midbrain of humans. Although these are the regions with the highest NM concentrations, it has been demonstrated that NM is also synthesized and accumulated in the neurons of other brain areas (Zecca et al. 2008, https://doi.org/10.1073/pnas.0808768105). Synthesis of peripheral melanins (e.g. in skin and hair) is mediated by tyrosinase, an enzyme also present at low levels in the brain. Whether brain tyrosinase may actually contribute to neuromelanin (NM) synthesis is currently unknown. Tyrosinase protein does not appear to be expressed in the SN (Ikemoto et al., 1998 [a study by Prof. Nagatsu]; https://doi.org/10.1016/S0304-3940(98)00649-1). We previously performed chemical analyses to elucidate the structure of NM in the SN (Wakamatsu et al. 2003, Wakamatsu et al. 2012, Zecca et al. 2008, https://doi.org/10.1073/pnas.0808768105), which suggested that the pigmented part of NM in the SN is derived from DA and CYS in a molar ratio of 2:1 (Zecca et al. 2008, https://doi.org/10.1073/pnas.0808768105; Wakamatsu et al. 2012, https://doi.org/10.1111/pcmr.12014). In addition, it was recently suggested that various catecholic metabolites are incorporated into NM from the SN and the LC, including DOPA, 3,4-dihydroxyphenylethanol (DOPE), 3,4-dihydroxyphenylethylene glycol (DOPEG), 3,4-dihydroxyphenylacetic acid (DOPAC) and 3,4-dihydroxymandelic acid (DOMA). These compounds are metabolites of DA and norepinephrine (NE) formed by oxidative deamination by monoamine oxidase followed by reduction/oxidation (Eisenhofer et al. 2004, https://pharmrev.aspetjournals.org/content/56/3/331.long; Wakamatsu et al. 2014, https://www.mdpi.com/1420-3049/19/6/8039; Wakamatsu et al. 2015, https://doi.org/10.1111/jnc.13237). The location of NM is related to the double-edged sword properties sometimes attributed to NM, as melanin formation in the cytosol of neurons could have cytotoxic properties. Once the NM polymer is formed, it can be a cytosolic scavenger for potentially neurotoxic sub-products, because Fe (III) or reactive oxygen species (ROS) oxidize catecholamine neurotransmitters. It has been reported that ?-synuclein could be one of the factors responsible for the positive association between PD and melanoma via its differential roles in melanin synthesis in melanoma and in dopaminergic neuronal cells (Goedert 2001, https://www.nature.com/articles/35081564; Pan et al. 2012, https://doi.org/10.1371/journal.pone.0045183). NM pigment accumulates inside specific autophagic organelles, which contain NM-iron complexes, along with lipids and various proteins (Zucca et al. 2017, https://doi.org/10.1016/j.pneurobio.2015.09.012). As NM–iron complexes are paramagnetic, they can be imaged using magnetic resonance imaging (MRI) (Sasaki et al. 2006, https://pubmed.ncbi.nlm.nih.gov/16837857/; Trujillo et al. 2017, https://doi.org/10.1002/mrm.26584; Sulzer et al. 2018, https://www.nature.com/articles/s41531-018-0047-3; Cassidy et al. 2019, https://www.pnas.org/content/116/11/5108.long). Cassidy et al. (2019) indicated that noninvasive NM-MRI is a promising tool that could have diverse research and clinical applications to investigate in vivo the role of DA in neuropsychiatric illness.
