The role of tyrosine hydroxylase as a key player in neuromelanin synthesis and the association of neuromelanin with Parkinson's disease.

The dark pigment neuromelanin (NM) is abundant in cell bodies of dopamine (DA) neurons in the substantia nigra (SN) and norepinephrine (NE) neurons in the locus coeruleus (LC) in the human brain. During the progression of Parkinson's disease (PD), together with the degeneration of the respective catecholamine (CA) neurons, the NM levels in the SN and LC markedly decrease. However, questions remain among others on how NM is associated with PD and how it is synthesized. The biosynthesis pathway of NM in the human brain has been controversial because the presence of tyrosinase in CA neurons in the SN and LC has been elusive. We propose the following NM synthesis pathway in these CA neurons: (1) Tyrosine is converted by tyrosine hydroxylase (TH) to L-3,4-dihydroxyphenylalanine (L-DOPA), which is converted by aromatic L-amino acid decarboxylase to DA, which in LC neurons is converted by dopamine ß-hydroxylase to NE; (2) DA or NE is autoxidized to dopamine quinone (DAQ) or norepinephrine quinone (NEQ); and (3) DAQ or NEQ is converted to eumelanic NM (euNM) and pheomelanic NM (pheoNM) in the absence and presence of cysteine, respectively. This process involves proteins as cysteine source and iron. We also discuss whether the NM amounts per neuromelanin-positive (NM+) CA neuron are higher in PD brain, whether NM quantitatively correlates with neurodegeneration, and whether an active lifestyle may reduce NM formation.

DOPA pheomelanin is increased in nigral neuromelanin of Parkinson's disease.

Neuromelanin (NM) in dopaminergic neurons of human substantia nigra (SN) has a melanic component that consists of pheomelanin and eumelanin moieties and has been proposed as a key factor contributing to dopaminergic neuron vulnerability in Parkinson's disease (PD). While eumelanin is considered as an antioxidant, pheomelanin and related oxidative stress are associated with compromised drug and metal ion binding and melanoma risk. Using postmortem SN from patients with PD or Alzheimer's disease (AD) and unaffected controls, we identified increased L-3,4-dihydroxyphenylalanine (DOPA) pheomelanin and increased ratios of dopamine (DA) pheomelanin markers to DA in PD SN compared to controls. Eumelanins derived from both DOPA and DA were reduced in PD group. In addition, we report an increase in DOPA pheomelanin relative to DA pheomelanin in PD SN. In AD SN, we observed unaltered melanin markers despite reduced DOPA compared to controls. Furthermore, synthetic DOPA pheomelanin induced neuronal cell death in vitro while synthetic DOPA eumelanin showed no significant effect on cell viability. Our findings provide insights into the different roles of pheomelanin and eumelanin in PD pathophysiology. We anticipate our study will lead to further investigations on pheomelanin and eumelanin individually as biomarkers and possibly therapeutic targets for PD.

Neuromelanins in brain aging and Parkinson's disease: synthesis, structure, neuroinflammatory, and neurodegenerative role.

Neuromelanins are compounds accumulating in neurons of human and animal brain during aging, with neurons of substantia nigra and locus coeruleus having the highest levels of neuromelanins. These compounds have melanic, lipid, peptide, and inorganic components and are contained inside special autolysosomes. Neuromelanins can participate in neuroprotective or toxic processes occurring in Parkinson's disease according to cellular environment. Their synthesis depends on the concentration of cytosolic catechols and is a protective process since it prevents the toxic accumulation of catechols-derived reactive compounds. Neuromelanins can be neuroprotective also by binding reactive/toxic metals to produce stable and non-toxic complexes. Extraneuronal neuromelanin released by dying dopamine neurons in Parkinson's disease activates microglia which generate reactive oxygen species, reactive nitrogen species, and proinflammatory molecules, thus producing still neuroinflammation and neuronal death. Synthetic neuromelanins have been prepared with melanic, protein structure, and metal content closely mimicking the natural brain pigment, and these models are also able to activate microglia. Neuromelanins have different structure, synthesis, cellular/subcellular distribution, and role than melanins of hair, skin, and other tissues. The main common aspect between brain neuromelanin and peripheral melanin is the presence of eumelanin and/or pheomelanin moieties in their structure.

Interactions of dopamine, iron, and alpha-synuclein linked to dopaminergic neuron vulnerability in Parkinson's disease and Neurodegeneration with Brain Iron Accumulation disorders.

Dopamine metabolism, alpha-synuclein pathology, and iron homeostasis have all been implicated as potential contributors to the unique vulnerability of substantia nigra dopaminergic neurons which preferentially decline in Parkinson's disease and some rare neurodegenerative disorders with shared pathological features. However, the mechanisms contributing to disease progression and resulting in dopaminergic neuron loss in the substantia nigra are still not completely understood. Increasing evidence demonstrates that disrupted dopamine, alpha-synuclein, and/or iron pathways, when combined with the unique morphological, physiological, and metabolic features of this neuron population, may culminate in weakened resilience to multiple stressors. This review analyzes the involvement of each of these pathways in dopamine neuron physiology and function, and discusses how disrupted interplay of dopamine, alpha-synuclein, and iron pathways may synergize to promote pathology and drive the unique vulnerability to disease states. We suggest that elucidating the interactions of dopamine with iron and alpha-synuclein, and the role of dopamine metabolism in driving pathogenic phenotypes will be critical for developing therapeutics to prevent progression in diseases that show degeneration of nigral dopamine neurons such as Parkinson's disease and the rare family of disorders known as Neurodegeneration with Brain Iron Accumulation.

Water-Soluble Melanin-Protein-Fe/Cu Conjugates Derived from Norepinephrine as Reliable Models for Neuromelanin of Human Brain Locus Coeruleus.

Water-soluble melanin-protein-Fe/Cu conjugates derived from norepinephrine and fibrillar ß-lactoglobulin are reliable models for neuromelanin (NM) of human brain locus coeruleus. Both iron and copper promote catecholamine oxidation and exhibit strong tendency to remain coupled in oligonuclear aggregates. The Fe-Cu clusters are EPR silent and affect the 1 H NMR spectra of the conjugates through a specific sequence of signals. Derivatives containing only Fe or Cu exhibit different NMR patterns. The EPR spectra show weak signals of paramagnetic FeIII in conjugates containing Fe or mixed Fe-Cu sites due to small amounts of mononuclear centers. The latter derivatives exhibit EPR signals for isolated CuII centers. These features parallel the EPR behavior of NM from locus coeruleus. The spectral data indicate that FeIII is bound to the melanic fraction, whereas CuII is bound on the protein fibrils, suggesting that the Fe-Cu clusters occur at the interface between the two components of the synthetic NMs.

Highly Effective Protocol for Differentiation of Induced Pluripotent Stem Cells (iPS) into Melanin-Producing Cells.

Melanin is a black/brown pigment present in abundance in human skin. Its main function is photo-protection of underlying tissues from harmful UV light. Natural sources of isolated human melanin are limited; thus, in vitro cultures of human cells may be a promising source of human melanin. Here, we present an innovative in vitro differentiation protocol of induced pluripotent stem cells (iPS) into melanin-producing cells, delivering highly pigmented cells in quantity and quality incomparably higher than any other methods previously described. Pigmented cells constitute over 90% of a terminally differentiated population and exhibit features characteristic for melanocytes, i.e., expression of specific markers such as MITF-M (microphthalmia-associated transcription factor isoform M), TRP-1 (tyrosinase-related protein 1), and TYR (tyrosinase) and accumulation of black pigment in organelles closely resembling melanosomes. Black pigment is unambiguously identified as melanin with features corresponding to those of melanin produced by typical melanocytes. The advantage of our method is that it does not require any sophisticated procedures and can be conducted in standard laboratory conditions. Moreover, our protocol is highly reproducible and optimized to generate high-purity melanin-producing cells from iPS cells; thus, it can serve as an unlimited source of human melanin for modeling human skin diseases. We speculate that FGF-8 might play an important role during differentiation processes toward pigmented cells.

Interaction of Neuromelanin with Xenobiotics and Consequences for Neurodegeneration; Promising Experimental Models.

Neuromelanin (NM) accumulates in catecholamine long-lived brain neurons that are lost in neurodegenerative diseases. NM is a complex substance made of melanic, peptide and lipid components. NM formation is a natural protective process since toxic endogenous metabolites are removed during its formation and as it binds excess metals and xenobiotics. However, disturbances of NM synthesis and function could be toxic. Here, we review recent knowledge on NM formation, toxic mechanisms involving NM, go over NM binding substances and suggest experimental models that can help identifying xenobiotic modulators of NM formation or function. Given the high likelihood of a central NM role in age-related human neurodegenerative diseases such as Parkinson's and Alzheimer's, resembling such diseases using animal models that do not form NM to a high degree, e.g., mice or rats, may not be optimal. Rather, use of animal models (i.e., sheep and goats) that better resemble human brain aging in terms of NM formation, as well as using human NM forming stem cellbased in vitro (e.g., mid-brain organoids) models can be more suitable. Toxicants could also be identified during chemical synthesis of NM in the test tube.

The neurobiology of human aggressive behavior: Neuroimaging, genetic, and neurochemical aspects.

In modern societies, there is a strive to improve the quality of life related to risk of crimes which inevitably requires a better understanding of brain determinants and mediators of aggression. Neurobiology provides powerful tools to achieve this end. Pre-clinical and clinical studies show that changes in regional volumes, metabolism-function and connectivity within specific neural networks are related to aggression. Subregions of prefrontal cortex, insula, amygdala, basal ganglia and hippocampus play a major role within these circuits and have been consistently implicated in biology of aggression. Genetic variations in proteins regulating the synthesis, degradation, and transport of serotonin and dopamine as well as their signal transduction have been found to mediate behavioral variability observed in aggression. Gene-gene and gene-environment interactions represent additional important risk factors for aggressiveness. Considering the social burden of pathological forms of aggression, more basic and translational studies should be conducted to accelerate applications to clinical practice, justice courts, and policy making.

Locus coeruleus imaging as a biomarker for noradrenergic dysfunction in neurodegenerative diseases.

Pathological alterations to the locus coeruleus, the major source of noradrenaline in the brain, are histologically evident in early stages of neurodegenerative diseases. Novel MRI approaches now provide an opportunity to quantify structural features of the locus coeruleus in vivo during disease progression. In combination with neuropathological biomarkers, in vivo locus coeruleus imaging could help to understand the contribution of locus coeruleus neurodegeneration to clinical and pathological manifestations in Alzheimer's disease, atypical neurodegenerative dementias and Parkinson's disease. Moreover, as the functional sensitivity of the noradrenergic system is likely to change with disease progression, in vivo measures of locus coeruleus integrity could provide new pathophysiological insights into cognitive and behavioural symptoms. Locus coeruleus imaging also holds the promise to stratify patients into clinical trials according to noradrenergic dysfunction. In this article, we present a consensus on how non-invasive in vivo assessment of locus coeruleus integrity can be used for clinical research in neurodegenerative diseases. We outline the next steps for in vivo, post-mortem and clinical studies that can lay the groundwork to evaluate the potential of locus coeruleus imaging as a biomarker for neurodegenerative diseases.

Iron, Myelin, and the Brain: Neuroimaging Meets Neurobiology.

Although iron is crucial for neuronal functioning, many aspects of cerebral iron biology await clarification. The ability to quantify specific iron forms in the living brain would open new avenues for diagnosis, therapeutic monitoring, and understanding pathogenesis of diseases. A modality that allows assessment of brain tissue composition in vivo, in particular of iron deposits or myelin content on a submillimeter spatial scale, is magnetic resonance imaging (MRI). Multimodal strategies combining MRI with complementary analytical techniques ex vivo have emerged, which may lead to improved specificity. Interdisciplinary collaborations will be key to advance beyond simple correlative analyses in the biological interpretation of MRI data and to gain deeper insights into key factors leading to iron accumulation and/or redistribution associated with neurodegeneration.

Neuromelanin-sensitive MRI as a noninvasive proxy measure of dopamine function in the human brain.

Neuromelanin-sensitive MRI (NM-MRI) purports to detect the content of neuromelanin (NM), a product of dopamine metabolism that accumulates with age in dopamine neurons of the substantia nigra (SN). Interindividual variability in dopamine function may result in varying levels of NM accumulation in the SN; however, the ability of NM-MRI to measure dopamine function in nonneurodegenerative conditions has not been established. Here, we validated that NM-MRI signal intensity in postmortem midbrain specimens correlated with regional NM concentration even in the absence of neurodegeneration, a prerequisite for its use as a proxy for dopamine function. We then validated a voxelwise NM-MRI approach with sufficient anatomical sensitivity to resolve SN subregions. Using this approach and a multimodal dataset of molecular PET and fMRI data, we further showed the NM-MRI signal was related to both dopamine release in the dorsal striatum and resting blood flow within the SN. These results suggest that NM-MRI signal in the SN is a proxy for function of dopamine neurons in the nigrostriatal pathway. As a proof of concept for its clinical utility, we show that the NM-MRI signal correlated to severity of psychosis in schizophrenia and individuals at risk for schizophrenia, consistent with the well-established dysfunction of the nigrostriatal pathway in psychosis. Our results indicate that noninvasive NM-MRI is a promising tool that could have diverse research and clinical applications to investigate in vivo the role of dopamine in neuropsychiatric illness.

Overexpression of Vesicular Monoamine Transporter-2 may Block Neurotoxic Metabolites from Cytosolic Dopamine: a Potential Neuroprotective Therapy for Parkinson's Disease.

The loss of nigrostriatal dopaminergic neurons containing neuromelanin underlies the motor symptoms of Parkinson's disease. Neuromelanin accumulation into autophagic lysosomes is evidence of ongoing cytosolic dopamine stress in these neurons during normal aging. The formation of neuromelanin is likely neuroprotective, as oxidation of cytosolic dopamine to quinones and aldehydes, as reviewed here, can produce a host of neurotoxic sequela. In addition to sequestration of dopamine and its metabolites in autophagic lysosomes, the uptake of dopamine into monoaminergic neurons mediated by vesicular monoamine transporter-2 (VMAT- 2), prevents dopamine oxidation. Dopamine is stable in monoaminergic vesicles due to their low pH, and thus overexpression of VMAT-2 may provide a target for potential neuroprotective therapy in Parkinson's disease.

Dopamine, Oxidative Stress and Protein-Quinone Modifications in Parkinson's and Other Neurodegenerative Diseases.

Dopamine (DA) is the most important catecholamine in the brain, as it is the most abundant and the precursor of other neurotransmitters. Degeneration of nigrostriatal neurons of substantia nigra pars compacta in Parkinson's disease represents the best-studied link between DA neurotransmission and neuropathology. Catecholamines are reactive molecules that are handled through complex control and transport systems. Under normal conditions, small amounts of cytosolic DA are converted to neuromelanin in a stepwise process involving melanization of peptides and proteins. However, excessive cytosolic or extraneuronal DA can give rise to nonselective protein modifications. These reactions involve DA oxidation to quinone species and depend on the presence of redox-active transition metal ions such as iron and copper. Other oxidized DA metabolites likely participate in post-translational protein modification. Thus, protein-quinone modification is a heterogeneous process involving multiple DA-derived residues that produce structural and conformational changes of proteins and can lead to aggregation and inactivation of the modified proteins.

Neuromelanin organelles are specialized autolysosomes that accumulate undegraded proteins and lipids in aging human brain and are likely involved in Parkinson's disease.

During aging, neuronal organelles filled with neuromelanin (a dark-brown pigment) and lipid bodies accumulate in the brain, particularly in the substantia nigra, a region targeted in Parkinson's disease. We have investigated protein and lipid systems involved in the formation of these organelles and in the synthesis of the neuromelanin of human substantia nigra. Membrane and matrix proteins characteristic of lysosomes were found in neuromelanin-containing organelles at a lower number than in typical lysosomes, indicating a reduced enzymatic activity and likely impaired capacity for lysosomal and autophagosomal fusion. The presence of proteins involved in lipid transport may explain the accumulation of lipid bodies in the organelle and the lipid component in neuromelanin structure. The major lipids observed in lipid bodies of the organelle are dolichols with lower amounts of other lipids. Proteins of aggregation and degradation pathways were present, suggesting a role for accumulation by this organelle when the ubiquitin-proteasome system is inadequate. The presence of proteins associated with aging and storage diseases may reflect impaired autophagic degradation or impaired function of lysosomal enzymes. The identification of typical autophagy proteins and double membranes demonstrates the organelle's autophagic nature and indicates that it has engulfed neuromelanin precursors from the cytosol. Based on these data, it appears that the neuromelanin-containing organelle has a very slow turnover during the life of a neuron and represents an intracellular compartment of final destination for numerous molecules not degraded by other systems.

A lysosome-plasma membrane-sphingolipid axis linking lysosomal storage to cell growth arrest.

Lysosomal accumulation of undegraded materials is a common feature of lysosomal storage diseases, neurodegenerative disorders, and the aging process. To better understand the role of lysosomal storage in the onset of cell damage, we used human fibroblasts loaded with sucrose as a model of lysosomal accumulation. Sucrose-loaded fibroblasts displayed increased lysosomal biogenesis followed by arrested cell proliferation. Notably, we found that reduced lysosomal catabolism and autophagy impairment led to an increase in sphingolipids ( i.e., sphingomyelin, glucosylceramide, ceramide, and the gangliosides GM3 and GD3), at both intracellular and plasma membrane (PM) levels. In addition, we observed an increase in the lysosomal membrane protein Lamp-1 on the PM of sucrose-loaded fibroblasts and a greater release of the soluble lysosomal protein cathepsin D in their extracellular medium compared with controls. These results indicate increased fusion between lysosomes and the PM, as also suggested by the increased activity of lysosomal glycosphingolipid hydrolases on the PM of sucrose-loaded fibroblasts. The inhibition of ß-glucocerebrosidase and nonlysosomal glucosylceramidase, both involved in ceramide production resulting from glycosphingolipid catabolism on the PM, partially restored cell proliferation. Our findings indicate the existence of a new molecular mechanism underlying cell damage triggered by lysosomal impairment.-Samarani, M., Loberto, N., Soldà, G., Straniero, L., Asselta, R., Duga, S., Lunghi, G., Zucca, F. A., Mauri, L., Ciampa, M. G., Schiumarini, D., Bassi, R., Giussani, P., Chiricozzi, E., Prinetti, A., Aureli, M., Sonnino, S. A lysosome-plasma membrane-sphingolipid axis linking lysosomal storage to cell growth arrest.

Neuromelanin detection by magnetic resonance imaging (MRI) and its promise as a biomarker for Parkinson's disease.

The diagnosis of Parkinson's disease (PD) occurs after pathogenesis is advanced and many substantia nigra (SN) dopamine neurons have already died. Now that therapies to block this neuronal loss are under development, it is imperative that the disease be diagnosed at earlier stages and that the response to therapies is monitored. Recent studies suggest this can be accomplished by magnetic resonance imaging (MRI) detection of neuromelanin (NM), the characteristic pigment of SN dopaminergic, and locus coeruleus (LC) noradrenergic neurons. NM is an autophagic product synthesized via oxidation of catecholamines and subsequent reactions, and in the SN and LC it increases linearly during normal aging. In PD, however, the pigment is lost when SN and LC neurons die. As shown nearly 25 years ago by Zecca and colleagues, NM's avid binding of iron provides a paramagnetic source to enable electron and nuclear magnetic resonance detection, and thus a means for safe and noninvasive measure in living human brain. Recent technical improvements now provide a means for MRI to differentiate between PD patients and age-matched healthy controls, and should be able to identify changes in SN NM with age in individuals. We discuss how MRI detects NM and how this approach might be improved. We suggest that MRI of NM can be used to confirm PD diagnosis and monitor disease progression. We recommend that for subjects at risk for PD, and perhaps generally for older people, that MRI sequences performed at regular intervals can provide a pre-clinical means to detect presymptomatic PD.

Interactions of iron, dopamine and neuromelanin pathways in brain aging and Parkinson's disease.

There are several interrelated mechanisms involving iron, dopamine, and neuromelanin in neurons. Neuromelanin accumulates during aging and is the catecholamine-derived pigment of the dopamine neurons of the substantia nigra and norepinephrine neurons of the locus coeruleus, the two neuronal populations most targeted in Parkinson's disease. Many cellular redox reactions rely on iron, however an altered distribution of reactive iron is cytotoxic. In fact, increased levels of iron in the brain of Parkinson's disease patients are present. Dopamine accumulation can induce neuronal death; however, excess dopamine can be removed by converting it into a stable compound like neuromelanin, and this process rescues the cell. Interestingly, the main iron compound in dopamine and norepinephrine neurons is the neuromelanin-iron complex, since neuromelanin is an effective metal chelator. Neuromelanin serves to trap iron and provide neuronal protection from oxidative stress. This equilibrium between iron, dopamine, and neuromelanin is crucial for cell homeostasis and in some cellular circumstances can be disrupted. Indeed, when neuromelanin-containing organelles accumulate high load of toxins and iron during aging a neurodegenerative process can be triggered. In addition, neuromelanin released by degenerating neurons activates microglia and the latter cause neurons death with further release of neuromelanin, then starting a self-propelling mechanism of neuroinflammation and neurodegeneration. Considering the above issues, age-related accumulation of neuromelanin in dopamine neurons shows an interesting link between aging and neurodegeneration.

Contrast mechanisms associated with neuromelanin-MRI.

PURPOSE: To investigate the physical mechanisms associated with the contrast observed in neuromelanin MRI. METHODS: Phantoms having different concentrations of synthetic melanins with different degrees of iron loading were examined on a 3 Tesla scanner using relaxometry and quantitative magnetization transfer (MT). RESULTS: Concentration-dependent T1 and T2 shortening was most pronounced for the melanin pigment when combined with iron. Metal-free melanin had a negligible effect on the magnetization transfer spectra. On the contrary, the presence of iron-laden melanins resulted in a decreased magnetization transfer ratio. The presence of melanin or iron (or both) did not have a significant effect on the macromolecular content, represented by the pool size ratio. CONCLUSION: The primary mechanism underlying contrast in neuromelanin-MRI appears to be the T1 reduction associated with melanin-iron complexes. The macromolecular content is not significantly influenced by the presence of melanin with or without iron, and thus the MT is not directly affected. However, as T1 plays a role in determining the MT-weighted signal, the magnetization transfer ratio is reduced in the presence of melanin-iron complexes. Magn Reson Med 78:1790-1800, 2017. © 2016 International Society for Magnetic Resonance in Medicine.

Elemental mapping of Neuromelanin organelles of human Substantia Nigra: correlative ultrastructural and chemical analysis by analytical transmission electron microscopy and nano-secondary ion mass spectrometry.

Neuromelanin (NM) is a compound which highly accumulates mainly in catecholamine neurons of the substantia nigra (SN), and is contained in organelles (NM-containing organelles) with lipid bodies and proteins. These neurons selectively degenerate in Parkinson's disease and NM can play either a protective or toxic role. NM-containing organelles of SN were investigated by Analytical Electron Microscopy (AEM) and Nano-Secondary Ion Mass Spectrometry (NanoSIMS) within human tissue sections with respect to ultrastructure and elemental composition. Within the NM-containing organelle, the single NM granules and lipid bodies had sizes of about 200-600 nm. Energy-Dispersive X-ray microanalysis spectra of the NM granules and lipid bodies were acquired with 100 nm beam diameter in AEM, NanoSIMS yielded elemental maps with a lateral resolution of about 150 nm. AEM yielded the quantitative elemental composition of NM granules and bound metals, e.g., iron with a mole fraction of about 0.15 atomic percent. Chemical analyses by AEM and NanoSIMS were consistent at the subcellular level so that nanoSIMS measurements have been quantitated. In NM granules of SN from healthy subjects, a significant amount of S, Fe, and Cu was found. In lipid bodies an amount of P consistent with the presence of phospholipids was measured. The improved detection limits of nanoSIMS offer new possibilities for chemical mapping, high-sensitivity trace element detection, and reduced acquisition times. Variations between individual NM granules can now be investigated effectively and quantitatively by NanoSIMS mapping Cu and Fe. This should yield new insight into the changes in chemical composition of NM pigments during healthy aging and disease. Neuromelanin-containing organelles of dopamine neurons in normal human substantia nigra were investigated by analytical electron mircoscopy and secondary ion mass spectroscopy (NanoSIMS) yielding the ultrastructure and elemental composition. In neuromelanin granules a significant amount of S, Fe and Cu was found. In lipid bodies an amount of P consistent with the presence of phospholipids was measured. The improved sensitivity of NanoSIMS shows differences in chemical composition between individual neuromelanin granules and allows to study chemical changes of neuromelanin organelles during aging and disease.