Immunocytochemical Quantification of Tyrosine Hydroxylase at a Cellular Level in the Mesencephalon of Control Subjects and Patients with Parkinson's and Alzheimer's Disease

Abstract: Parkinson's disease is characterized by massive degeneration of the melanized dopaminergic neurons in the substantia nigra. The functional capacity of the surviving nigral neurons is affected, as indicated by the subnormal levels of tyrosine hydroxylase (TH) mRNA in these neurons and the presence in the parkinsonian mesencephalon of melanized neurons lacking TH immunoreactivity. This is apparently in contradiction with the known overactivity of dopamine synthesis and release that occurs in the remaining dopaminergic terminals. To test the ability of the surviving neurons to express TH protein, a semiquantitative immunocytochemical method was developed. The relative amounts of TH were estimated with a computer-assisted image analysis system in the dopaminergic neurons of representative mesencephalic sections of control and parkinsonian brains and for comparison in brains from patients with Alzheimer's disease. In control brains, the mean TH content per neuron differed from one subject to another and between the different dopaminergic cell groups of the mesencephalon in the same subject. Within a given dopaminergic region, the level of TH was variable among neurons. In patients with Parkinson's disease, the ratio of TH protein content per neuron in the substantia nigra by reference to that of the central gray substance was reduced. In patients with Alzheimer's disease, the amount of TH was selectively reduced in the remaining dopaminergic neurons of the ventral tegmental area, a region characterized by a loss in dopaminergic neurons. The decrease in cellular TH content might therefore be related to the presence of the neurodegenerative process in the area considered. In patients with Parkinson's disease, the incapacity of the surviving neurons to express normal TH levels may reduce the efficiency of the hyperactivity mechanisms that develop in the remaining striatal dopaminergic terminals.     

Modifications of the iron-neuromelanin system in Parkinson's disease

Parkinson's disease is a common neurodegenerative disorder with a mainly sporadic aetiology, although a number of monogenic familiar forms are known. Most of the motor symptoms are due to selective depletion of dopaminergic, neuromelanin-containing neurones of the substantia nigra pars compacta. Neuromelanin is the dark insoluble macromolecule that confers the black (substantia nigra) or grey (locus coeruleus) colour to monoaminergic basal ganglia. In particular, nigral neurones are pigmented because of the accumulation of by-products of oxidative metabolism of the neurotransmitter dopamine. The occurrence of dopamine (and all the enzymatic machinery required for dopamine synthesis, re-uptake and disposal) and neuromelanin, and a large amount of iron ions that interact with them, makes dopaminergic nigral neurones peculiarly susceptible to oxidative stress conditions that, in turn, may become amplified by the iron-neuromelanin system itself. In this mini-review we describe biophysical evidence for iron-neuromelanin modifications that support this hypothesis. Furthermore, we discuss the formation of the covalent linkage between alpha-synuclein and neuromelanin from the early stages of the disease.     

Botulinum A Like Type B and Tetanus Toxins Fulfils Criteria for Being a Zinc-Dependent Protease

Abstract: Iron is abnormally accumulated in the substantia nigra pars compacta of patients with Parkinson's disease (PD). Because neuronal and glial iron uptake seems to be mediated by the binding of ferrotransferrin to a specific high-affinity receptor on the cell surface, the number of transferrin receptors could be altered in this disease. The regional distribution of specific binding sites for human ¹²⁵I-diferric transferrin has been studied in the mesencephalon, on cryostat-cut sections from autopsy brains of control subjects and parkinsonian patients by in vitro autoradiography. Densities of binding sites were highest in the central gray substance (˜10 fmol/mg of tissue equivalent), intermediate in the catecholaminergic cell group A8, superior colliculus, and ventral tegmental area, and almost nonexistent in the substantia nigra. The density of ¹²⁵I-transferrin binding sites was not significantly different between parkinsonian and control brains in any region analyzed. These results show that in the mesencephalon the regional density of transferrin binding sites is lowest in the dopaminergic cell groups, which are the most vulnerable to PD, and suggest that iron does not accumulate through an increased density of transferrin receptors at the level of the substantia nigra.     

Magnetic investigations of human mesencephalic neuromelanin

Pigmentation of neurons in substantia nigra is due to neuromelanin, a pigment that stores large amounts of iron. Human mesencephalic neuromelanin has been investigated by means of magnetic susceptibility measurements as a function of temperature. Magnetic measurements provide a physico-chemical characterization of the iron cluster buried in the organic melanin matrix and support the view that iron is not simply chelated, but rather is organized in a three-dimensional network. The paramagnetism of isolated iron ions is observed, in agreement with electron paramagnetic resonance spectroscopy. Furthermore, antiferromagnetic grains with a large size distribution function are present. These grains contain N spins coupled antiferromagnetically; however, N(1/2) spins are decoupled from the grain bulk and parallelly aligned. The latter subgrains are superparamagnetic with a blocking temperature ranging between 5 K and room temperature. This behavior has not been observed in synthetic melanin, where the paramagnetic contribution is strongly enhanced. Preliminary results on pigment isolated from patients affected by Parkinson's disease, a neurodegenerative pathology involving primarily pigmented neurons in substantia nigra pars compacta, show a lower total magnetization compared to control neuromelanin. The temperature behavior of zero field cooling and field cooling magnetizations is similar for both. The significant depletion of iron content in Parkinson's disease neuromelanin could indicate a progressive Fe migration from its storage environment to the cytosol.     

Is there a rationale for neuroprotection against dopamine toxicity in Parkinson's disease?

Parkinson's disease is a progressive neurological disease caused by rather selective degeneration of the dopaminergic neurons in the substantia nigra. Though subject to intensive research, the etiology of this nigral loss is still undetermined and treatment is basically symptomatic. The current major hypothesis is that nigral neuronal death in PD is due to excessive oxidative stress generated by auto and enzymatic oxidation of the endogenous neurotransmitter dopamine (DA), the formation of neuromelanin (NM) and the presence of a high concentration of iron. In this review article although we concisely describe the effects of NM and iron on neuronal survival, we mainly focus on the molecular mechanisms of DA-induced apoptosis. DA exerts its toxic effects through its oxidative metabolites either in vitro or in vivo The oxidative metabolites then activate a very intricate web of signals, which culminate in cell death. The signal transduction pathways and genes, which are associated with DA toxicity are described in detail.     

Residual substantia nigra neuromelanin in Parkinson's disease is cross-linked to alpha-synuclein

The pigmentation of substantia nigra pars compacta dopaminergic neurons is due to the presence of neuromelanin, an irregular macromolecular pigment belonging to the family of melanins. Depletion of neuromelanin in Parkinson's disease is typically indicated by loss of brown color in this area. Unlike that from controls, the pigment extracted from substantia nigra of parkinsonian patients seems to be mainly composed by highly cross-linked, protease-resistant proteic material and the neuromelanin macromolecule appears to be a minor presence. In the present paper we describe the isolation by SDS-PAGE of this proteic component after cleavage of the melanin backbone under solubilizing conditions. A single band is observed, which has been identified as alpha-synuclein by western blotting. As expected, the same process performed on a control specimen did not show occurrence of any major proteic component. Nevertheless, extraction from a 91 years old control with Lewy bodies displayed minor alpha-synuclein immunoreactive aggregates, whereas inclusion of free alpha-synuclein was not observed at all. Results reported here support the view that alpha-synuclein accumulates within substantia nigra neurons and is entrapped in pigment granules during neuromelanin biosynthesis, i.e. before the melanin depletion characteristic of Parkinson's disease starts.     

Identification and kainic acid-induced up-regulation of low-affinity p75 neurotrophin receptor (p75NTR) in the nigral dopamine neurons of adult rats

Parkinson's disease is a common and severe debilitating neurological disease that results from massive and progressive degenerative death of dopamine neurons in the substantia nigra, but the mechanisms of neuronal degeneration and disease progression remains largely obscure. We are interested in possible implications of low-affinity p75 neurotrophin receptor (p75NTR), which may mediate neuronal apoptosis in the central nervous system, in triggering cell death of the nigral dopamine neurons. The RT-PCR and immunohistochemistry were carried out to detect if p75NTR is expressed in these nigral neurons and up-regulated by kainic acid (KA) insult in adult rats. It revealed p75NTR-positive immunoreactivity in the substantia nigra, and co-localization of p75NTR and tyrosine hydroxylase (TH) was found in a large number of substantia nigra neurons beside confirmation of p75NTR in the choline acetyltransferase (ChAT)-positive forebrain neurons. Cell count data further indicated that about 47-100% of TH-positive nigral neurons and 98-100% of ChAT-positive forebrain neurons express p75NTR. More interestingly, significant increasing in both p75NTR mRNA and p75NTR-positive neurons occurred rapidly following KA insult in the substantia nigra of animal model. The present study has provided first evidence on p75NTR expression and KA-inducing p75NTR up-regulation in substantia nigra neurons in rodent animals. Taken together with previous data on p75NTR functions in neuronal apoptosis, this study also suggests that p75NTR may play important roles in neuronal cell survival or excitotoxic degeneration of dopamine neurons in the substantia nigra in pathogenesis of Parkinson's disease in human beings.     

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.     

Chemical neurotransmission in the parkinsonian brain

In Parkinson's disease the progressive loss of nigrostriatal dopamine neurons leads to striatal dopamine deficiency and correlates with the severity of parkinsonian disability. The findings concerning dopamine receptors both in vitro and in vivo are not consistent, possibly reflecting differences in patient populations, but the presynaptic defect in dopaminergic neurotransmission is greater than that seen in postsynaptic receptor binding studies. The cholinergic neurons in the extrapyramidal nuclei are relatively well preserved, but subcortico-cortical and -hippocampal cholinergic neurons degenerate in relation to the degree of dementia. The decreased GABA receptor binding in the parkinsonian substantia nigra possibly reflects the loss of nigral dopamine neurons, since nigral GABA receptors are located on these neurons. Of the various neuropeptides, the concentration of met- and leu-enkephalin seems to be reduced in the striatum. In the substantia nigra the concentration of substance P decreases, together with the met-enkephalin and cholecystokinin levels. The concentration of somatostatin decreases in the frontal cortex and hippocampus of demented patients. With the exception of the association between cortical somatostatin deficiency and intellectual deterioration, the role of the neuropeptides in the pathophysiology and clinical features of Parkinson's disease are not yet fully understood.     

Iron as a vulnerability factor in nigrostriatal degeneration in aging and Parkinson's disease.

Recent findings strengthen the connection between iron accumulation in the basal ganglia, oxidative stress and nigrostriatal degeneration. Oxidative stress appears to be elevated in the normal human substantia nigra in comparison with other brain regions, and further increases occur in Parkinson's disease. Accumulation of iron may contribute to degeneration of nigral dopamine neurons by catalyzing oxidative damage to cell components and also by perturbing the network of interactions that modulate cellular redox status.     

The relevance of iron in the pathogenesis of Parkinson's disease

Alterations of iron levels in the brain has been observed and documented in a number of neurodegenerative disorders including Parkinson's disease (PD). The elevated nigral iron levels observed in PD may reflect a dysfunction of brain iron homeostasis. Under normal physiological conditions excess iron can be sequestrated in ferritin and neuromelanin. Alternatively, the excess iron may represent a component of brain iron deposition associated with ageing. The aetiology of idiopathic PD largely remains an enigma. However, intensive investigations have provided a host of putative mechanisms that might contribute to the pathogenesis underlying the characteristic degeneration of the dopaminergic neurons in the substantia nigra (SN). The mechanisms proposed include oxidative (and nitrative) stress, inflammation, excitotoxicity, mitochondrial dysfunction, altered proteolysis and finally apoptotic induced cell death. Iron-mediated cellular destruction is mediated primarily via reactive oxygen or/and nitrogen species induced oxidative stress. Furthermore, these pathogenic mechanisms appear to be closely interlinked to the cascade of events leading to cellular death. There are conflicting reports about the stage during disease progression at which nigral iron change occurs in PD. Some have found that there are no changes in iron content SN in asymptomatic incidental Lewy body disease, suggesting it may represent a secondary event in the cascade of neuronal degeneration. In contrast, others have found an elevation of iron in SN in pre-clinical stages. These discrepancies may be attributed to the occurrence of different sub-groups of the disease. This concurs with the notion that PD represents a group of related diseases with a number of potential pathogenic pathways.     

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.     

Neuromelanin associated redox-active iron is increased in the substantia nigra of patients with Parkinson's disease

Degeneration of dopaminergic neurones during Parkinson's disease is most extensive in the subpopulation of melanized-neurones located in the substantia nigra pars compacta. Neuromelanin is a dark pigment produced in the dopaminergic neurones of the human substantia nigra and has the ability to bind a variety of metal ions, especially iron. Post-mortem analyses of the human brain have established that oxidative stress and iron content are enhanced in association with neuronal death. As redox-active iron (free Fe2+ form) and other transition metals have the ability to generate highly reactive hydroxyl radicals by a catalytic process, we investigated the redox activity of neuromelanin (NM)-aggregates in a group of parkinsonian patients, who presented a statistically significant reduction (- 70%) in the number of melanized-neurones and an increased non-heme (Fe3+) iron content as compared with a group of matched-control subjects. The level of redox activity detected in neuromelanin-aggregates was significantly increased (+ 69%) in parkinsonian patients and was highest in patients with the most severe neuronal loss. This change was not observed in tissue in the immediate vicinity of melanized-neurones. A possible consequence of an overloading of neuromelanin with redox-active elements is an increased contribution to oxidative stress and intraneuronal damage in patients with Parkinson's disease.     

Transition Metals, Ferritin, Glutathione, and Ascorbic Acid in Parkinsonian Brains

The regional distributions of iron, copper, zinc, magnesium, and calcium in parkinsonian brains were compared with those of matched controls. In mild Parkinson's disease (PD), there were no significant differences in the content of total iron between the two groups, whereas there was a significant increase in total iron and iron (III) in substantia nigra of severely affected patients. Although marked regional distributions of iron, magnesium, and calcium were present, there were no changes in magnesium, calcium, and copper in various brain areas of PD. The most notable finding was a shift in the iron (II)/iron (III) ratio in favor of iron (III) in substantia nigra and a significant increase in the iron (III)-binding, protein, ferritin. A significantly lower glutathione content was present in pooled samples of putamen, globus pallidus, substantia nigra, nucleus basalis of Meynert, amygdaloid nucleus, and frontal cortex of PD brains with severe damage to substantia nigra, whereas no significant changes were observed in clinicopathologically mild forms of PD. In all these regions, except the amygdaloid nucleus, ascorbic acid was not decreased. Reduced glutathione and the shift of the iron (II)/iron (III) ratio in favor of iron (III) suggest that these changes might contribute to pathophysiological processes underlying PD.     

Neuromelanin can protect against iron-mediated oxidative damage in system modeling iron overload of brain aging and Parkinson's disease

In Parkinson's disease (PD), dopamine neurons containing neuromelanin selectively degenerate. Neuromelanin binds iron and accumulates in aging. Iron accumulates in reactive form during aging, PD, and is involved in neurodegeneration. It is not clear how the interaction of neuromelanin and iron can be protective or toxic by modulating redox processes. Here, we investigated the interaction of neuromelanin from human substantia nigra with iron in the presence of ascorbic acid, dopamine, and hydrogen peroxide. We observed that neuromelanin blocks hydroxyl radical production by Fenton's reaction, in a dose-dependent manner. Neuromelanin also inhibited the iron-mediated oxidation of ascorbic acid, thus sparing this major antioxidant molecule in brain. The protective effect of neuromelanin on ascorbate oxidation occurs even in conditions of iron overload into neuromelanin. The blockade of iron into a stable iron–neuromelanin complex prevents dopamine oxidation, inhibiting the formation of neurotoxic dopamine quinones. The above processes occur intraneuronally in aging and PD, thus showing that neuromelanin is neuroprotective. The iron–neuromelanin complex is completely decomposed by hydrogen peroxide and its degradation rate increases with the amount of iron bound to neuromelanin. This occurs in PD when extraneuronal iron–neuromelanin is phagocytosed by microglia and iron–neuromelanin degradation releases reactive/toxic iron.     

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.     

Increased Protein Oxidation in Human Substantia Nigra Pars Compacta in Comparison with Basal Ganglia and Prefrontal Cortex Measured with an Improved Dinitrophenylhydrazine Assay

Abstract: The dopaminergic phenotype of neurons in human substantia nigra deteriorates during normal aging, and loss of these neurons is prominent in Parkinson's disease. These degenerative processes are hypothesized to involve oxidative stress. To compare oxidative stress in the nigra and related regions, we measured carbonyl modifications of soluble proteins in postmortem samples of substantia nigra, basal ganglia, and prefrontal cortex from neurologically normal subjects, using an improved 2,4-dinitrophenylhydrazine assay. The protein carbonyl content was found to be about twofold higher in substantia nigra pars compacta than in the other regions. To further analyze this oxidative damage, the distribution of carbonyl groups on soluble proteins was determined by western immunoblot analysis. This method revealed that carbonyl content of the major proteins in each region was linearly dependent on molecular weight. This distribution raises the possibility that protein carbonyl content is controlled by a size-dependent mechanism in vivo. Our results suggest that oxidative stress is elevated in human substantia nigra pars compacta in comparison with other regions and that oxidative damage is higher within the dopaminergic neurons. Elevated oxidative damage may contribute to the degeneration of nigral dopaminergic neurons in aging and in Parkinson's disease.     

Signal alterations of the basal ganglia in the differential diagnosis of Parkinson’s disease: a retrospective case-controlled MRI data bank analysis

Background

Based upon the acquainted loss of dopaminergic neurons in the substantia nigra in Parkinson’s disease (PD), we hypothesised changes in magnetic resonance imaging signal intensities of the basal ganglia to be useful as an additional technical tool in the diagnostic work-up.

Methods

Region-of-interest analyses (substantia nigra and globus pallidus internus) of T2-weighted scans were performed in seventy subjects with PD, 170 age- and gender-matched controls and 38 patients with an atypical form of neurodegenerative Parkinsonian syndrome (N?=?11 multisystem atrophy, N?=?22 progressive supranuclear palsy, N?=?5 corticobasal syndrome).

Results

In patients with PD, significant changes in signal intensities within the substantia nigra were observed compared to controls at p?<?0.001. For the globus pallidus internus, signal alterations in PD and progressive supranuclear palsy were found to be significant (p?<?0.001) if compared to controls. Furthermore, signal changes of substantia nigra correlated with signal intensities of globus pallidus internus in the ipsilateral hemisphere in both groups. Sensitivity was 86% and specificity was 90% for the combined analysis of substantia nigra and globus pallidus internus in the complete patient sample versus controls.

Conclusions

Signal alterations of substantia nigra and globus pallidus internus in routine magnetic resonance imaging were useful to distinguish patients with PD from controls. In addition, signal changes in globus pallidus internus could be used to differentiate progressive supranuclear palsy patients from controls. These analyses have the potential to serve as an additional non-invasive technical tool to support the individual differential diagnosis of PD.