Iron and Other Metals in Neuromelanin, Substantia Nigra, and Putamen of Human Brain

Abstract: Radiochemical neutron activation analysis has been used to determine the concentration of 36 elements in neuromelanin, 22 elements in substantia nigra, and 32 elements in putamen of healthy subjects without signs of neurological disorders. Substantia nigra and putamen tissues were carefully dissected from the brain using special surgical instruments and tools as well as an adequate sampling procedure to avoid the risk of metal contamination during sampling. Neuromelanin was isolated from putamen by a multiple-step procedure (extraction with phosphate buffer, lipid and protein elimination by methanol extraction, and sodium dodecyl sulfate-proteinase). The isolated pigment as well as substantia nigra and putamen underwent neutron activation analysis involving irradiation in a high-neutron-flux reactor, radiochemical separations, and counting of the induced radionuclides by computer-based γ-ray spectrometry. Iron was the element present in the highest concentration in all analyzed samples. The amount of iron was similar in substantia nigra and putamen (3,000 and 3,830 ng/mg wet weight, respectively) and 10 times higher in neuromelanin (30,800 ng/mg dry weight). Zinc was also present at high levels in three samples, ranging from 16.8 (substantia nigra) to 1,500 ng/mg (neuromelanin). Elements such as Zn, Cr, Se, Sr, Co, Sb, Ni, Hg, Ce, Au, Ag, Ta, and Sc were present in neuromelanin at much higher concentrations than in substantia nigra and putamen. These findings indicate that substantia nigra and putamen contain metals at higher concentrations than observed in blood and that neuromelanin has a particular affinity for metals.     

Basal Lipid Peroxidation in Substantia Nigra Is Increased in Parkinson''s Disease

Polyunsaturated fatty acid (PUFA) levels (an index of the amount of substrate available for lipid peroxidation) were measured in several brain regions from patients who died with Parkinson's disease and age-matched control human postmortem brains. PUFA levels were reduced in parkinsonian substantia nigra compared to other brain regions and to control tissue. However, basal malondialdehyde (MDA; an intermediate in the lipid peroxidation process) levels were increased in parkinsonian nigra compared with other parkinsonian brain regions and control tissue. Expressing basal MDA levels in terms of PUFA content, the difference between parkinsonian and control substantia nigra was even more pronounced. Stimulating MDA production by incubating tissue with FeSO4 plus ascorbic acid, FeSO4 plus H2O2, or air alone produced lower MDA levels in the parkinsonian substantia nigra, probably reflecting the lower PUFA content. These results may indicate that an increased level of lipid peroxidation continues to occur in the parkinsonian nigra up to the time of death, perhaps because of continued exposure to excess free radicals derived from some endogenous or exogenous neurotoxic species.     

Selective Increase of Iron in Substantia Nigra Zona Compacta of Parkinsonian Brains

Histochemical and biochemical determinations of total iron, iron (II), and iron (III) contents in brain regions from Parkinson's and Alzheimer's diseases have demonstrated a selective increase of total iron content in parkinsonian substantia nigra zona compacta but not in the zona reticulata. The increase of iron content is mainly in iron (III). The ratio of iron (II):iron (III) in zona compacta changes from almost 2:1 to 1:2. This change is thought to be relevant and may contribute to the selective elevation of basal lipid peroxidation in substantia nigra reported previously. Iron may be available in a free state and thus can participate in autooxidation of dopamine with the resultant generation of H2O2 and oxygen free radicals.     

Oxidative DNA Damage in the Parkinsonian Brain: An Apparent Selective Increase in 8-Hydroxyguanine Levels in Substantia Nigra

Abstract: Oxidative damage has been implicated in the pathology of Parkinson's disease (PD), e.g., rises in the level of the DNA damage product, 8-hydroxy-2'-deoxyguanosine, have been reported. However, many other products result from oxidative DNA damage, and the pattern of products can be diagnostic of the oxidizing species. Gas chromatography/mass spectrometry was used to examine products of oxidation and deamination of all four DNA bases in control and PD brains. Products were detected in all brain regions examined, both normal and PD. Analysis showed that levels of 8-hydroxyguanine (8-OHG) tended to be elevated and levels of 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FAPy guanine) tended to be decreased in PD. The most striking difference was a rise in 8-OHG in PD substantia nigra ( p = 0.0002); rises in other base oxidation/deamination products were not evident, showing that elevation in 8-OHG is unlikely to be due to peroxynitrite (ONOO⁻) or hydroxyl radicals (OH^•), or to be a prooxidant effect of treatment with l -Dopa. However, some or all of the rise in 8-OHG could be due to a change in 8-OHG/FAPy guanine ratios rather than to an increase in total oxidative guanine damage.     

A Selective Increase in Particulate Superoxide Dismutase Activity in Parkinsonian Substantia Nigra

The total activity of superoxide dismutase (SOD) and cytosolic and particulate activity of SOD in human substantia nigra and cerebellum were measured by a spectrophotometric method based on the ability of SOD to inhibit the autoxidation of adrenaline. The cytosolic and particulate isoenzymes of SOD were differentiated by the inclusion of potassium cyanide which selectively inhibits cytosolic copper/zinc-dependent SOD activity. In autopsied human brains, there was no difference in total SOD activity, or the activity of SOD in cytosol in substantia nigra of patients dying with Parkinson's disease compared to age-matched controls. However, the activity of the particulate form of SOD was higher in the parkinsonian substantia nigra compared to control tissue. In the cerebellum there was no difference in the total, cytosolic, or particulate activity of SOD between parkinsonian patients and age-matched controls. Increased activity of SOD in particulate fraction may be a protective response to elevated levels of toxic free radicals in the parkinsonian substantia nigra. Alternatively, increased SOD activity may induce cell death through the accumulation of hydrogen peroxide.     

Increased Nigral Iron Content and Alterations in Other Metal Ions Occurring in Brain in Parkinson's Disease

Levels of iron, copper, zinc, manganese, and lead were measured by inductively coupled plasma spectroscopy in parkinsonian and age-matched control brain tissue. There was 31-35% increase in the total iron content of the parkinsonian substantia nigra when compared to control tissue. In contrast, in the globus pallidus total iron levels were decreased by 29% in Parkinson's disease. There was no change in the total iron levels in any other region of the parkinsonian brain. Total copper levels were reduced by 34-45% in the substantia nigra in Parkinson's disease; no difference was found in the other brain areas examined. Zinc levels were increased in substantia nigra in Parkinson's disease by 50-54%, and the zinc content of the caudate nucleus and lateral putamen was also raised by 18-35%. Levels of manganese and lead were unchanged in all areas of the parkinsonian brain studied when compared to control brains, except for a small decrease (20%) in manganese content of the medial putamen. Increased levels of total iron in the substantia nigra may cause the excessive formation of toxic oxygen radicals, leading to dopamine cell death.     

Activation of Caspase-3 in Developmental Models of Programmed Cell Death in Neurons of the Substantia Nigra

Programmed cell death has been proposed to play a role in the death of neurons in acute and chronic degenerative neurologic disease. There is now evidence that the caspases, a family of cysteine proteases, mediate programmed cell death in various cells. In neurons, caspase-3 (CPP32/Yama/apopain), in particular, has been proposed to play a role. We examined the expression of caspase-3 in three models of programmed cell death affecting neurons of the substantia nigra in the rat: natural developmental neuron death and induced developmental death following either striatal target injury with quinolinic acid or dopamine terminal lesion with intrastriatal injection of 6-hydroxydopamine. Using an antibody to the large (p17) subunit of activated caspase-3, we have found that activated enzyme is expressed in apoptotic profiles in all models. Increased p17 immunostaining correlated with increased enzyme activity. The subcellular distribution of activated caspase-3 differed among the models: In natural cell death and the target injury model, it was strictly nuclear, whereas in the toxin model, it was also cytoplasmic. We conclude that p17 immunostaining is a useful marker for programmed cell death in neurons of the substantia nigra.     

Mössbauer Spectroscopic Studies of Purified Human Neuromelanin Isolated from the Substantia Nigra

Abstract: ⁵⁷Fe Mössbauer spectroscopy at different temperatures has been used to characterize the nature of purified human neuromelanin isolated from the substantia nigra. The quantitative determination of iron(III) by estimation of the overall area of the Mössbauer spectrum at room temperature reveals an iron content of 2.8 ± 1.4%. No subspectra corresponding to divalent iron could be observed in these spectra. The derived Mössbauer parameters lead to the conclusion that the iron sites in the human neuromelanin are similar to those of human hemosiderin (or ferritin). However, owing to the water insolubility of the purified neuromelanin, it must be concluded that the neuromelanin hemosiderin (or ferritin) is bound in a protein matrix that makes it insoluble and difficult to stain histochemically. This protein attachment to neuromelanin is important in that it is what makes it different from synthetic dopamine melanin.     

Increased Iron in the Substantia Nigra Compacta of the MPTP-Lesioned Hemiparkinsonian African Green Monkey: Evidence from Proton Microprobe Elemental Microanalysis

Abstract: The association of free radicals and particularly free iron in the pathogenesis of idiopathic Parkinson's disease and MPTP-induced parkinsonism remains controversial. Whereas the actual cause of dopamine cell death in the substantia nigra compacta (SNc) remains unknown, disturbances in lipid peroxidation and subsequent mitochondrial and cell membrane disruption has been demonstrated. In a genetically susceptible host, abnormal elimination of oxygen and trace metal free radicals may further damage dopamine cells. Using a unilaterally MPTP-treated African Green monkey, which showed obvious contralateral hemiparkinsonism, the total free iron concentration was measured. Iron, Fe²⁺ and Fe³⁺, but not other trace elements, was significantly elevated in the SNc compared with the opposite unlesioned side, which was similar to separate control animals. Iron content in the SNc, periaqueductal gray area, and crus cerebri was 228–270 ppm. Normal control SNc was 285 (±59) ppm, whereas iron levels of 532 (±151) ppm were found in the MPTP-lesioned SNc. These animals were drug naive and not on long-term levodopa maintenance. Proton microprobe elemental analysis was matched against adjacent immunocytochemically stained tissue slices to ensure the cells studied were in the SNc. Iron was found not only in the degenerating dopamine cells themselves but also in the surrounding matrix and glial cells. Whether free iron that is not bound to neuromelanin is responsible for dopamine cell death as suggested by these experiments remains to be proved.     

Interaction of human substantia nigra neuromelanin with lipids and peptides

Neuromelanin was isolated from human substantia nigra using different procedures. In the pigment isolated by any of these procedures a peptide component covalently bound to the melanic structure was found, as shown by treatment with reagents known to eliminate noncovalently bound proteins. The amino acid content of such a peptide component was reproducible and corresponded to approximately 15% of the neuromelanin weight. Neuromelanin also showed the ability to absorb specifically lipid molecules, approximately 20% of its weight, and among these lipids cholesterol was identified, constituting approximately 5% of the total lipid mixture. A synthetic melanin, incubated with putamen homogenate, bound tissue peptides with an amino acid content quite close to that of neuromelanin. The same synthetic melanin adsorbed a lower amount of lipids from the putamen homogenate compared with neuromelanin. The sulfur content of neuromelanin was also reproducible even using different isolation procedures. A nonpigmented tissue like corpus callosum was used as a control and extracted by the method used for neuromelanin isolation; a total elimination of tissue components was found, thus demonstrating the capability of the reported procedures to isolate neuromelanin alone. The presence of a peptide component in the neuromelanin structure and the selective affinity for lipid molecules suggest new aspects of the functional role and metabolic pathway of neuromelanin.     

Substantia Nigra γ-Aminobutyric Acid Receptors in Huntington''s Disease

The specific binding of [3H]gamma-aminobutyric acid (GABA) to nigral GABA receptors has been studied in postmortem brains from controls and patients with Huntington's disease (HD). A specific increase in the number of high-affinity binding sites for [3H]GABA was observed in HD patients, analogous to changes observed in rat substantia nigra [3H]GABA binding after striatal kainic acid (KA) lesion. The results provide further support for the striatal KA lesion in the rat as an animal model of HD. The implications of the results for the proposed therapeutic potential of GABA agonists in HD are discussed.     

Purified Human Neuromelanin, Synthetic Dopamine Melanin as a Potential Model Pigment, and the Normal Human Substantia Nigra: Characterization by Electron Paramagnetic Resonance Spectroscopy

Abstract: Neuromelanin is a poorly understood pigment that accumulates in catecholaminergic neurons during normal aging. Electron paramagnetic resonance spectroscopy, an especially effective technique for investigating melanins, is used in the present study to show unambiguously that neuromelanin is a melanin; however, it is not well modeled by synthetic dopamine melanin and thus is an atypical melanin. Some of the unusual features of neuromelanin can be explained by postulating two distinct sources for its free radicals, the dominant one possibly derived from a precursor containing sulfur. Examination of human substantia nigra by electron paramagnetic resonance spectroscopy during the purification of neuromelanin also demonstrates, contrary to some other studies, that a portion of the paramagnetic metal ions in this tissue are bound to the pigment in situ. Combined with previous histochemical data, these observations have implications for the mechanism through which neuromelanin accumulates in vivo and are consistent with its having a cytoprotective function under normal conditions, but a cytotoxic role at advanced ages and in patients with Parkinson's disease. Other results of this study show that homogenizing tissues during the purification of any natural pigment may cause contamination of the pigment by extraneous metal ions and that subsequent incubation in hot acid, though most effective in removing metal ions and hydrolyzing proteins, leads to degradation of melanin. A purification procedure using incubation in acid at room temperature, however, is well suited for identifying and characterizing unknown natural pigments by electron paramagnetic resonance spectroscopy.     

Decreased Ferritin Levels in Brain in Parkinson''s Disease

Ferritin levels were measured in postmortem brain tissue from patients dying with Parkinson's disease [treated with L-3,4-dihydroxyphenylalanine (L-DOPA)] and from control patients. Ferritin levels were decreased in the substantia nigra, caudate-putamen, globus pallidus, cerebral cortex, and cerebellum when compared with age-matched control tissues. However, in CSF from L-DOPA-treated patients and in serum from L-DOPA-treated and untreated parkinsonian patients, ferritin levels were normal. Previous studies have suggested an increased total iron content in substantia nigra of parkinsonian brain. The failure of substantia nigra ferritin formation to be stimulated by increased iron levels suggests some defect in iron handling in this critical brain region in Parkinson's disease. The reason for decreased ferritin levels throughout the parkinsonian brain is not clear but does not seem to reflect a general system deficit in ferritin.     

Enkephalinergic Markers in Substantia Nigra and Caudate Nucleus from Parkinsonian Subjects

Marked reductions in opiate receptor binding (-42%), "enkephalinase" activity (-39%), and Met5-enkephalin levels (-72%) accompanied the well-established dopamine depletion in the substantia nigra pars compacta of Parkinsonian subjects. In contrast, enkephalinergic markers were not significantly modified in caudate nucleus.     

Evidence for a Glycidic-Lipidic Matrix in Human Neuromelanin, Potentially Responsible for the Enhanced Iron Sequestering Ability of Substantia Nigra

Abstract: The high-resolution solid-state ¹³C-NMR spectrum of a neuromelanin specimen (from patients dying from nonneurological diseases) is compared with that obtained from enzymatically prepared dopamine-melanin. The main differences between the two spectra suggest the occurrence in neuromelanin of a glycidic/lipidic matrix tightly associated with the melanin macromolecule. Atomic emission spectroscopy revealed high iron content (1.5%) in the neuromelanin specimen, in full agreement with previous reports. These observations support the view that neuromelanin acts as a strong chelating (and insolubilizing) system for iron ions and further suggest that the attack to this compact composite substrate may be an important step to allow the release of iron ions responsible for the increased lipid peroxidation reported in the pathogenesis of Parkinson's disease.     

Iron-Melanin Complex in Substantia Nigra of Parkinsonian Brains: An X-Ray Microanalysis

Using energy-dispersive x-ray analysis on an electron microscope working in the scanning transmission electron microscopy mode equipped with a microanalysis system, we studied the subcellular distribution of trace elements in neuromelanin-containing neurons of the substantia nigra zona compacta (SNZC) of three cases of idiopathic Parkinson's disease (PD) [one with Alzheimer's disease (AD)] and of three controls, in Lewy bodies of SNZC, and in synthetic dopamine-melanin chemically charged or uncharged with Fe. Weak but significant Fe peaks similar to those of a synthetic melanin-Fe3+ complex were seen only in intraneuronal highly electron-dense neuromelanin granules of SNZC cells of PD brains, with the highest levels in a case of PD plus AD, whereas a synthetic melanin-Fe2+ complex showed much lower iron peaks, indicating that neuromelanin has higher affinity for Fe3+ than for Fe2+. No detectable Fe was seen in nonmelanized cytoplasm of SNZC neurons and in the adjacent neuropil in both PD and controls, in Lewy bodies in SNZC neurons in PD, and in synthetic dopamine-melanin uncharged with iron. These findings, demonstrating for the first time a neuromelanin-iron complex in dopaminergic SNZC neurons in PD, support the assumption that an iron-melanin interaction contributes significantly to dopaminergic neurodegeneration in PD and PD plus AD.     

Biochemistry of Somatodendritic Dopamine Release in Substantia Nigra: An In Vivo Comparison with Striatal Dopamine Release

Abstract: The somatodendritic release of dopamine in substantia nigra previously has been suggested to be nonvesicular in nature and thus to differ from the classical, exocytotic release of dopamine described for the dopaminergic nerve terminal in striatum. We have compared the effects of reserpine, a compound that disrupts vesicular sequestration of monoamines, on the storage and release of dopamine in substantia nigra and striatum of rats. Reserpine administration (5 mg/kg, i.p.) significantly decreased the tissue level of dopamine in substantia nigra pars reticulata, substantia nigra pars compacta, and striatum. In these brain areas, reserpine-induced reductions in tissue dopamine level occurred within 2 h and persisted at 24 h postdrug. In vivo measurements using microdialysis revealed that reserpine administration rapidly decreased the extracellular dopamine concentration to nondetectable levels in substantia nigra as well as in striatum. In both structures, it was observed that reserpine treatment significantly attenuated the release of dopamine evoked by a high dose of amphetamine (10 mg/kg, i.p.) given 2 h later. In contrast, dopamine efflux in response to a low dose of amphetamine (2 mg/kg, i.p.) was not altered by reserpine pretreatment either in substantia nigra or in striatum. The present data suggest the existence, both at the somatodendritic and at the nerve terminal level, of a vesicular pool of dopamine that is the primary site of transmitter storage and that can be displaced by high but not low doses of amphetamine. The physiological release of dopamine in substantia nigra and in striatum is dependent on the integrity of this vesicular store.     

Glial Cell Line-Derived Neurotrophic Growth Factor Inhibits Apoptotic Death of Postnatal Substantia Nigra Dopamine Neurons in Primary Culture

Abstract: Glial cell line-derived neurotrophic factor (GDNF) was identified on the basis of its ability to enhance the development of embryonic mesencephalic dopamine neurons. It remains unknown whether GDNF is a physiologically relevant trophic factor for these neurons. We have shown that natural cell death among dopamine neurons of the substantia nigra occurs largely postnatally. To investigate whether GDNF may have the ability to support these neurons during their period of natural cell death, we have used a postnatal primary culture model. We find that GDNF is able to support the viability of postnatal nigral dopamine neurons by inhibiting apoptotic death. This ability of GDNF shows both regional specificity for the nigra and cellular specificity for the dopamine phenotype. Among eight other neurotrophic factors previously reported to support embryonic dopamine neurons, GDNF was unique in this ability. Thus, GDNF meets this criterion for a physiologically relevant trophic factor for dopamine neurons of the substantia nigra.     

Synthesis and Release of Dopamine in Rat Brain: Comparison Between Substantia Nigra Pars Compacta, Pars Reticulata, and Striatum

Dopamine (DA) is synthesized and released not only from the terminals of the nigrostriatal dopaminergic neuronal pathway, but also from the dendrites in the substantia nigra. We have investigated the regulation of the DA turnover, the DA synthesis rate, and the DA release in the substantia nigra pars compacts (SNpc) and pars reticulata (SNpr) in vivo. As a measure of DA turnover, we have assessed the concentrations of 3,4-dihydroxyphenylacetic acid and homovanillic acid. As a measure of the DA synthesis rate, we have determined the 3,4-dihydroxyphenylalanine accumulation after inhibition of aromatic L-amino acid decarboxylase by 3-hydroxybenzylhydrazine. As a measure of DA release, we have investigated the disappearance rate of DA after inhibition of its synthesis by alpha-methyl-p-tyrosine and the 3-methoxytyramine accumulation following monoamine oxidase inhibition by pargyline. Both the DA turnover and the DA synthesis rate increased following treatment with the DA receptor antagonist haloperidol and decreased following treatment with the DA receptor agonist apomorphine in the SNpc and in the SNpr, but the effects of the drugs were less pronounced than in the striatum. gamma-Butyrolactone treatment, which suppresses the firing of the dopaminergic neurons, increased the DA synthesis rate in the striatum (165%), but had no such effect in the SNpc or SNpr. Haloperidol, apomorphine, and gamma-butyrolactone increased, decreased, and abolished, respectively, the DA release in the striatum, but the drugs had no or only slight effects on the alpha-methyl-p-tyrosine-induced DA disappearance and on the pargyline-induced 3-methoxytyramine accumulation in the SNpc or SNpr. Taken together, these results indicate that the DA synthesis rate, but not the DA release, are influenced by DA receptor activity and neuronal firing in the SNpc and SNpr. This is in contrast to the situation in the striatum, where both the DA synthesis rate and the DA release are under such control.