Investigation of the lipid component of neuromelanin

Summary. Objective: Neuromelanin (NM) is different to other melanins in that its ultrastructure includes a lipid component. The objectives of this study were to identify and quantify lipids associated with NM. Results: Quantification of the lipid component associated with the pigment on electron micrographs demonstrated that this component comprises 35% of the NM granule volume in the normal brain. The irregular ultrastructural appearance of the NM granules was quite different to the round regular boundary of melanin granules. Using reversed phase high performance liquid chromatography (HPLC) coupled with atmospheric pressure chemical ionization (APCI) mass spectrometry we demonstrated that the isoprenoid dolichol accounted for approximately 12% of total NM pigment mass. Low levels of other lipids were detectable (cholesterol, ubiquinone-10 and α-tocopherol) and account for <0.05% of NM lipid, in contrast to cholesterol accounting for 35% of total brain lipids. Conclusion: Unlike other melanins, a substantial proportion of NM volume is comprised of lipid and the major type of lipid associated with NM granules is the isoprenoid dolichol.     

Decreased nigral neuromelanin in Alzheimer's disease

Using manual morphometric techniques, we estimated the amount of neuromelanin in hematoxylin and eosin-stained sections of the pars compacta of the substantia nigra of 19 Alzheimer's patients without nigral Lewy bodies and 12 age-matched controls. Our estimates showed that the mean area and areal fraction of neuromelanin were lower in Alzheimer's disease than controls but the number and size of the neuronal cell bodies, nuclei and nucleoli did not differ between the two groups. We speculated that the decreased amount of neuromelanin in nigral neuronal cell bodies could have resulted from neurofibrillary degeneration, retrograde degeneration from damage of nigral dopaminergic terminals in the striatum by the beta amyloid protein of the diffuse plaques and possibly transneuronal degeneration from damage of cell bodies or dendrites of nigral neurons by their plaque- and tangle-ravaged striatal, neocortical and other subcortical nigral connections. We hypothesized that any or all of the above types of degeneration could have lowered the rate of dopamine metabolism and the formation of one of its by-products, neuromelanin. Our study shows that a decrease in the amount of histopathologically-observable nigral neuromelanin commonly occurs in Alzheimer's disease without nigral Lewy bodies.     

Lipid content determines aggregation of neuromelanin granules in vitro

The neuromelanin pigment of the substantia nigra of the human brain is closely associated with lipids and other non-melanogenic compounds which appear to contribute to the unique and complex morphology of neuromelanin pigment granules. In this work we show that insoluble granules isolated from the human substantia nigra associate in vitro to form pigment aggregates similar to those present in the human brain. Extraction of neuromelanin-associated polar lipids by methanol and/or hexane significantly enhanced melanin aggregate size. A marked (10-fold) increase in granule size was seen after methanol treatment, whereas the application of hexane after methanol reduced this pro-aggregation effect. We have previously reported that hexane and methanol remove the neuromelanin-associated polyisoprenoids dolichol and cholesterol respectively. Thus, the current data suggests that pigment-associated lipids may be a factor regulating pigment aggregation and neuromelanin granule size in vivo.     

Synthetic neuromelanin is toxic to dopaminergic cell cultures

Summary. In the present study, primary cultures of mesencephalic dopaminergic cells were exposed to synthetic dopamine neuromelanin (NM) for 48 hrs at concentrations of 0, 1, 10, 20, 50 and 100 μg NM/ml medium. Differently prepared synthetic NM with or without incorporated iron and NM oxidatively damaged by hydrogen peroxide were used. All NMs affected cellular structures e.g. as swelling of neural processes, rounding of cells, and occasional inclusion of neuromelanin particles. Cell numbers were uniformly and dose dependently reduced. Exposure to MPP⁺ and ferric iron led to cytotoxic changes which could be further aggravated by oxidatively damaged NM, suggesting cytotoxicity of soluble compounds of NM in predamaged neurons. Received February 4, 2002; accepted February 27, 2002     

Functional effects of neuromelanin and synthetic melanin in model systems

Summary. The function of the dark polymer pigment neuromelanin found in catecholaminergic neurons of the human brain is not understood, especially as most published data are based upon a synthetic model melanin which differs structurally to the native pigment. Nevertheless human neuromelanin has been shown to efficiently bind transition metals such as iron, as well as other potentially toxic molecules. The pigment may have a protective function in the healthy brain by, for example, contributing to iron homeostasis within pigmented nuclei. We have demonstrated that synthetic dopamine melanin stimulates cell damage in both cell lines and primary cells in vitro, an effect associated with increased hydroxyl radical production and apoptosis. In contrast, at low iron concentrations native neuromelanin does not induce cell damage but rather protects cells in culture from oxidative stress. This protective function appears to be lost at high iron concentrations where neuromelanin saturated with iron functions as a source of oxidative load, rather than an iron chelator. Changes to neuromelanin and tissue iron load in Parkinson’s disease may decrease the protective potential of the pigment, thus increase the potential for cell damage in this disorder.     

Evidence for specific phases in the development of human neuromelanin

Summary. Neuromelanin is a dark-coloured pigment which forms in the dopamine neurons of the human midbrain. Here we describe the age-related development and regulation of neuromelanin within these dopamine neurons. 10 μm sections from formalin-fixed midbrain from 29 people spanning the ages of 24 weeks to 95 years old were either stained with a basic Nissl substance stain (0.5% cresyl violet), or processed unstained. After locating the substantia nigra using the stained sections, digital photos were taken of individual ventral substantia nigra neurons in the unstained sections, and the cellular area occupied by pigment, and optical density were measured using computer software. These measurements demonstrated three developmental phases. Neuromelanin was not present at birth and initiation of pigmentation began at approximately 3 years of age, followed by a period of increasing pigment granule number and increasing pigment granule colouration until age 20. In middle and later life the colour of the pigment granules continued to darken but was not associated with any substantial growth in pigment volume. The identification of three phases and changes in the rate of neuromelanin production over time suggests the regulation of neuromelanin production and turnover, possibly through enzymatic processes.     

Structural investigations of neuromelanin by pyrolysis-gas chromatography/mass spectrometry

Summary. Pyrolysis combined with gas chromatography and mass spectrometry (Py-GC/MS) was applied for structural investigations of the human substantia nigra neuromelanin. Using synthetic neuromelanins, we have demonstrated that Py-GC/MS is suitable for identification and differentiation of both eumelanin (dopamine-derived) and pheomelanin (cysteinyldopamine-derived) component of the pigment. Structural information on melanin monomers was inferred from their pyrolytic markers. When the human neuromelanin was subjected to pyrolysis, none of the heterocyclic, sulfur-containing markers of pheomelanin component was detected among the thermal degradation products. We have concluded that nigral pigment isolated from normal brain tissue does not contain benzothiazine-type monomers, and that cysteinyldopamine-originated units may be incorporated into the polymer in uncyclized form. The most abundant pyrolysis product was identified as limonene, which indicates that nigral pigment is tightly associated with an isoprenoid-type compound. Pyrolysis in the presence of the methylating reagent allowed identification of high levels of saturated and monounsaturated straight-chain C14–C18 fatty acid species chemically bound to the pigment macromolecule.     

Lack of toxicity of human neuromelanin to rat brain dopaminergic neurons

Neuromelanin extracts from the substantia nigra of a parkinsonian and a non-parkinsonian case were injected into the substantia nigra and caudate-putamen of rat brains. Rats were sacrificed at periods ranging from 3 days to 8 months. Substantial amounts of neuromelanin remained at both injection sites even after 8 months, indicating a very slow rate of phagocytosis. No differences were seen in the reaction to control or parkinsonian melanin, and no neurotoxicity attributable to the melanin was observed. These data fail to support the hypothesis of neurotoxicity of melanin as a cause of Parkinson's disease.     

The neuromelanin of human substantia nigra and its interaction with metals

Summary. Neuromelanin (NM) is a peculiar biochemical component of several neurons in the Substantia Nigra (SN), the target area of the degenerative process in Parkinson Disease (PD). SN NM has peculiarities as to its composition and an impressive capacity of chelating metals, iron in particular, but not exclusively. Gaining insights into the structural and functional characteristics of NM should help understanding the reasons of selective vulnerability of nigral neurons in many parkinsonian conditions. From the present data a protective role of NM can be postulated until the buffering capability toward heavy metals are exhausted. The overloading of NM with iron and other metals in neurons may trigger inflammatory and degenerative processes aggravating the underlying pathological condition. Received December 19, 2001; accepted January 28, 2002     

Total and paramagnetic metals in human substantia nigra and its neuromelanin

Summary A number of hypotheses on the etiology of Parkinson's disease and other CNS disorders postulate a role of metal ions and/or neuromelanin. As part of an investigation of the interactions between neuromelanin and metal ions, we have studied the amount and type of metal ions in human neuromelanin in intact substantia nigra and in isolated neuromelanin using electron paramagnetic resonance (EPR), which selectively measures metal ions which are in valence states that have unpaired electrons and total reflection X-ray fluorescence (TXRF), which measures total metals. EPR also is a principal technique for studying the biophysics of melanins by analysis of its free radicals. The studies of substantia nigra with TXRF indicated the presence of substantial amounts of iron, zinc, lead, copper, maganese, and titanium at concentrations up to 4 times greater than those of non-pigmented brain tissue (basis pedunculi). The concentrations of metal ions in isolated neuromelanin were 5–260 times higher than in substantia nigra. The studies with EPR indicated that there were substantial amounts of paramagnetic metals ions, especially iron, bound to neuromelanin in intact substantia nigra, and the presence of these metal ions modified the EPR spectra of the free radicals of neuromelanin. We conclude: 1. Compared to other regions of the mid-brain, the substantia nigra contains increased amounts of many different metal ions; 2. Many of these metal ions are in paramagnetic valence states; 3. There are high concentrations of paramagnetic metal ions bound to neuromelanin. These results are consistent with the hypotheses that postulate a role of metal ions in promoting oxidative reactions in pigmented neurons.     

Acid fast staining of oxidized neuromelanin and lipofuscin in the human brain.

Acid fast staining of the bleached residuum of substantia nigra neuromelanin and of oxidized inferior olive lipofuscin was demonstrated in paraffin and frozen sections stained with the acetic acid, carbol fuchsin method of Barbeito-Lopez and the aldehyde fuchsin method of Gomori. Acid fast staining occurred when sections were exposed to a prestain oxidation with potassium permanganate in conjunction with a poststain differentiation with dilute hydrochloric acid. The acid fast staining with acetic acid, carbol fuchsin was differentiable and in contrast to the acid fast staining with aldehyde fuchsin which was nondifferentiable. A possible histochemical basis for differentiable and nondifferentiable acid fast staining was discussed. The identify of the bleached residuum of neuromelanin as lipofuscin was also discussed.     

Neuroaxonal dystrophy with neuromelanin deposition,neurofibrillary tangles,and neuronal loss

Neuroaxonal spheroids became evident microscopically after the autopsy of a 45-year-old woman with pigmentation of the globus pallidus suggesting Hallervorden-Spatz disease. In our opinion the fine floccular pigment seen electron-microscopically in many of the axonal spheroids is melanin, an end product of catecholamine metabolism. Neurofibrillary degeneration, senile plaques, and granulovacuolar degeneration in the hippocampus produced a picture of Alzheimer's disease. Pontocerebellar degeneration and motor neuron disease were also observed.     

Reduced signal of locus ceruleus in depression in quantitative neuromelanin magnetic resonance imaging

We used a neuromelanin-magnetic resonance imaging technique to investigate abnormalities in the locus ceruleus in depression. We examined 20 patients with major depression and 43 age-matched controls using a 3 T scanner with a neuromelanin-sensitive sequence. The signal intensities of the areas corresponding to the rostral, middle, and caudal portions of the locus ceruleus were measured, and the contrast ratio relative to the adjacent pontine tegmentum was calculated. In controls, the contrast ratio in the middle portion was higher than in the rostral and caudal areas. In patients, contrast ratios in the rostral and middle portions were significantly decreased in comparison with controls, suggesting dysfunction of the ascending noradrenergic system. Neuromelanin-magnetic resonance imaging can be used to visualize abnormalities in the locus ceruleus of depressive patients.     

The neuromelanin of human substantia nigra: structure,synthesis and molecular behaviour

The pigmented neurons of the substantia nigra (SN) are typically lost in Parkinson's disease: however the possible relationship between neuronal vulnerability and the presence of neuromelanin (NM) has not been elucidated. Early histological studies revealed the presence of increasing amounts of NM in the SN with aging in higher mammals, showed that NM granules are surrounded by membrane, and comparatively evaluated the pigmentation of SN in different animal species. Histochemical studies showed the association of NM with lipofuscins. However, systematic investigations of NM structure, synthesis and molecular interactions have been undertaken only during the last decade. In these latter studies, NM was identified as a genuine melanin with a strong chelating ability for iron and affinity for compounds such as lipids, pesticides, and MPP+. The affinity of NM for a variety of inorganic and organic toxins is consistent with a postulated protective function for NM. Moreover, the neuronal accumulation of NM during aging, and the link between its synthesis and high cytosolic concentration of catechols suggests a protective role. However, its putative neuroprotective effects could be quenched in conditions of toxin overload.