Dopamine and vesicular monoamine transport loss supports incidental Lewy body disease as preclinical idiopathic Parkinson.

Incidental Lewy body disease (ILBD) is a neuropathological diagnosis of brains with Lewy bodies without clinical neuropsychiatric symptoms. Dopaminergic deficits suggest a relationship to preclinical Parkinson's disease (PD). We now report a subregional pattern of striatal dopamine loss in ILBD cases, with dopamine found significantly decreased in the putamen (-52%) and only to a lower extent in the caudate (-38%, not statistically significant); this is similar to the pattern in idiopathic PD in various neurochemical and in vivo imaging studies. We aimed to find out if our recently reported impaired storage of dopamine in striatal synaptic vesicles prepared from striatal tissue of cases with idiopathic PD might be an early or even causative event. We undertook parallel measurements of [3H]dopamine uptake and vesicular monoamine transporter (VMAT)2 binding sites by the specific label [3H]dihydrotetrabenazine on vesicular preparation from caudate and putamen in ILBD. Neither specific uptake of dopamine and binding of [3H]dihydrotetrabenazine, nor mean values of the calculated ratios of dopamine uptake and VMAT2 binding, a measure of uptake rate per transport site, were significantly different between ILBD and controls. ATP-dependence of [3H]dopamine uptake revealed significantly higher rates in putamen than in caudate at saturating concentrations of ATP in controls, a subregional difference lost in ILBD. Our findings support a loss of the normally higher VMAT2 activity in putamen as a contributing factor to the higher susceptibility of the putamen to dopamine depletion in idiopathic PD. Moreover, we suggest ILBD postmortem tissue as a valuable source for testing hypotheses on processes in idiopathic PD.

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.

Onset and mortality of Parkinson's disease in relation to type II diabetes.

OBJECTIVES: There is growing evidence that Parkinson's disease and diabetes are partially related diseases; however, the association between the two, and the impact of specific treatments, are still unclear. We evaluated the effect of T2D and antidiabetic treatment on age at PD onset and on all-cause mortality. RESEARCH DESIGN AND METHODS: The standardized rate of T2D was calculated for PD patients using the direct method and compared with subjects with essential tremor (ET) and the general Italian population. Age at onset and survival were also compared between patients without T2D (PD-noT2D), patients who developed T2D before PD onset (PD-preT2D) and patients who developed T2D after PD onset (PD-postT2D). RESULTS: We designed a retrospective and prospective study. The T2D standardized ratio of PD (N = 8380) and ET (N = 1032) patients was 3.8% and 6.1%, respectively, while in the Italian general population, the overall prevalence was 5.3%. In PD-preT2D patients, on antidiabetic treatment, the onset of PD was associated with a + 6.2 year delay (p < 0.001) while no difference was observed in PD-postT2D. Occurrence of T2D before PD onset negatively affected prognosis (adjusted hazard ratio = 1.64 [95% CI 1.33-2.02]; p < 0.001), while no effect on survival was found in PD-postT2D subjects (hazard ratio = 0.86, [95% CI 0.53-1.39]; p = 0.54). CONCLUSIONS: T2D, treated with any antidiabetic therapy before PD, is associated with a delay in its onset. Duration of diabetes increases mortality in PD-preT2D, but not in PD-postT2D. These findings prompt further studies on antidiabetic drugs as a potential disease-modifying therapy 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.

Age-associated insolubility of parkin in human midbrain is linked to redox balance and sequestration of reactive dopamine metabolites.

The mechanisms by which parkin protects the adult human brain from Parkinson disease remain incompletely understood. We hypothesized that parkin cysteines participate in redox reactions and that these are reflected in its posttranslational modifications. We found that in post mortem human brain, including in the Substantia nigra, parkin is largely insoluble after age 40 years; this transition is linked to its oxidation, such as at residues Cys95 and Cys253. In mice, oxidative stress induces posttranslational modifications of parkin cysteines that lower its solubility in vivo. Similarly, oxidation of recombinant parkin by hydrogen peroxide (H2O2) promotes its insolubility and aggregate formation, and in exchange leads to the reduction of H2O2. This thiol-based redox activity is diminished by parkin point mutants, e.g., p.C431F and p.G328E. In prkn-null mice, H2O2 levels are increased under oxidative stress conditions, such as acutely by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine toxin exposure or chronically due to a second, genetic hit; H2O2 levels are also significantly increased in parkin-deficient human brain. In dopamine toxicity studies, wild-type parkin, but not disease-linked mutants, protects human dopaminergic cells, in part through lowering H2O2. Parkin also neutralizes reactive, electrophilic dopamine metabolites via adduct formation, which occurs foremost at the primate-specific residue Cys95. Further, wild-type but not p.C95A-mutant parkin augments melanin formation in vitro. By probing sections of adult, human midbrain from control individuals with epitope-mapped, monoclonal antibodies, we found specific and robust parkin reactivity that co-localizes with neuromelanin pigment, frequently within LAMP-3/CD63+ lysosomes. We conclude that oxidative modifications of parkin cysteines are associated with protective outcomes, which include the reduction of H2O2, conjugation of reactive dopamine metabolites, sequestration of radicals within insoluble aggregates, and increased melanin formation. The loss of these complementary redox effects may augment oxidative stress during ageing in dopamine-producing cells of mutant PRKN allele carriers, thereby enhancing the risk of Parkinson's-linked neurodegeneration.

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.

COVID-19 and possible links with Parkinson's disease and parkinsonism: from bench to bedside.

This Viewpoint discusses insights from basic science and clinical perspectives on coronavirus disease 2019 (COVID-19)/severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection in the brain, with a particular focus on Parkinson's disease. Major points include that neuropathology studies have not answered the central issue of whether the virus enters central nervous system neurons, astrocytes or microglia, and the brain vascular cell types that express virus have not yet been identified. Currently, there is no clear evidence for human neuronal or astrocyte expression of angiotensin-converting enzyme 2 (ACE2), the major receptor for viral entry, but ACE2 expression may be activated by inflammation, and a comparison of healthy and infected brains is important. In contrast to the 1918 influenza pandemic and avian flu, reports of encephalopathy in COVID-19 have been slow to emerge, and there are so far no documented reports of parkinsonism apart from a single case report. We recommend consensus guidelines for the clinical treatment of Parkinson's patients with COVID-19. While a role for the virus in causing or exacerbating Parkinson's disease appears unlikely at this time, aggravation of specific motor and non-motor symptoms has been reported, and it will be important to monitor subjects after recovery, particularly for those with persisting hyposmia.

Evidence for Dopamine Abnormalities in the Substantia Nigra in Cocaine Addiction Revealed by Neuromelanin-Sensitive MRI.

OBJECTIVE: Recent evidence supports the use of neuromelanin-sensitive MRI (NM-MRI) as a novel tool to investigate dopamine function in the human brain. The authors investigated the NM-MRI signal in individuals with cocaine use disorder, compared with age- and sex-matched control subjects, based on previous imaging studies showing that this disorder is associated with blunted presynaptic striatal dopamine. METHODS: NM-MRI and T1-weighted images were acquired from 20 participants with cocaine use disorder and 35 control subjects. Diagnostic group effects in NM-MRI signal were determined using a voxelwise analysis within the substantia nigra. A subset of 20 cocaine users and 17 control subjects also underwent functional MRI imaging using the monetary incentive delay task, in order to investigate whether NM-MRI signal was associated with alterations in reward processing. RESULTS: Compared with control subjects, cocaine users showed significantly increased NM-MRI signal in ventrolateral regions of the substantia nigra (area under the receiver operating characteristic curve=0.83). Exploratory analyses did not find a significant correlation of NM-MRI signal to activation of the ventral striatum during anticipation of monetary reward. CONCLUSIONS: Given that previous imaging studies show decreased dopamine signaling in the striatum, the finding of increased NM-MRI signal in the substantia nigra provides additional insight into the pathophysiology of cocaine use disorder. One interpretation is that cocaine use disorder is associated with a redistribution of dopamine between cytosolic and vesicular pools, leading to increased accumulation of neuromelanin. The study findings thus suggest that NM-MRI can serve as a practical imaging tool for interrogating the dopamine system in addiction.

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.

Neuronal Proteins as Targets of 3-Hydroxykynurenine: Implications in Neurodegenerative Diseases.

The neurotoxic activity of the tryptophan metabolite 3-hydroxykynurenine (3OHKyn) in neurodegenerative disorders, such as Parkinson's and Alzheimer's diseases, is related to oxidative stress and 3OHKyn interaction with cellular proteins. The pattern of protein modification induced by 3OHKyn involves the nucleophilic side chains of Cys, His, and Lys residues, similarly to the one promoted by dopamine and other catecholamines. In the present work, we have analyzed the reactivity of 3OHKyn toward the neuronal targets α-synuclein (and its N-terminal fragments 1-6 and 1-15) and amyloid-ß peptides (1-16 and 1-28) and characterized the resulting conjugates through spectrometric (LC-MS/MS) and spectroscopic (UV-vis, fluorescence, NMR) techniques. The amino acid residues of α-synuclein and amyloid-ß peptides involved in derivatizations by 3OHKyn and its autoxidation products (belonging to the xanthommatin family) are Lys and His, respectively. The pattern of protein modification is expanded in the conjugates obtained in the presence of the metal ions copper(II) or iron(III), reflecting a more oxidizing environment that in addition to adducts with protein/peptide residues also favors the fragmentation of the protein. These results open the perspective to using the 3OHKyn-protein/peptide synthetic conjugates to explore their competence to activate microglia cell cultures as well as to unravel their role in neuroinflammatory conditions.

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.