Substantia Nigra Of Human Brain Research Articles

Gender and age-related differences in morphometric characteristics of neurons in human brain substantia nigra

Introduction. Age-related morphological changes in the brain and those taking place in Parkinson disease (PD) are similar in their nature but differ in intensity. Quantitative evaluation of the neurons’ characteristics in substantia nigra pars compacta (SNc) in men and women during aging will allow to use obtained values as a reference while studying PD. Objective: to study gender- and age-related morphometric characteristics of neurons in SNc of the human brain. Materials and methods. Morphometric evaluation of SNc neurons in autopsy human brain specimens (n=12) of normal aging men and women (aged 52–87 years) was performed. The sections were stained with cresyl violet and for thyrosine hydroxylase (dopamine marker). A total number of neurons and number of dopaminergic neurons in particular in ventral and dorsal regions of SNc were counted; cellular and nuclear size was also estimated. Results. In the aging brain, the most pronounced morphological changes occur in the medial, lateral, and intermediate segments of the ventral region of SNc. In the medial segment, the overall neuronal density was decreased by 33%, while in the lateral and intermediate segments of the ventral region of SNc it was decreased by 23%. In the medial and in the lateral and intermediate segments, density of the dopaminergic neurons was decreased by 28% and 24% respectively. Survived neurons showed increased cellular size and reduced nuclear size. In women, basic morphometric characteristics of neurons in the lateral and intermediate segments of the ventral region of SNc were higher than in men. Conclusions. In normal aging, involution of the brain structures in SNc is more pronounced in its ventral region. Interestingly, involution occurs more slowly in a female brain than in a male brain.

Synthesis, Structure Characterization, and Evaluation in Microglia Cultures of Neuromelanin Analogues Suitable for Modeling Parkinson’s Disease

In the substantia nigra of human brain, neuromelanin (NM) released by degenerating neurons can activate microglia with consequent neurodegeneration, typical of Parkinson's disease (PD). Synthetic analogues of NM were prepared to develop a PD model reproducing the neuropathological conditions of the disease. Soluble melanin-protein conjugates were obtained by melanization of fibrillated β-lactoglobulin (fLG). The melanic portion of the conjugates contains either eumelanic (EufLG) or mixed eumelanic/pheomelanic composition (PheofLG), the latter better simulating natural NMs. In addition, the conjugates can be loaded with controlled amounts of iron. Upon melanization, PheofLG-Fe conjugates maintain the amyloid cross-β protein core as the only structurally organized element, similarly to human NMs. The similarity in composition and structural organization with the natural pigment is reflected by the ability of synthetic NMs to activate microglia, showing potential of the novel conjugates to model NM induced neuroinflammation. Thus, synthetic NM/microglia constitute a new model to develop anti-Parkinson drugs.

Intranuclear ubiquitin-immunopositive structures in human substantia nigra neurons

Marinesco bodies were discovered in substantia nigra neurons of human brain in 1902. The relationships between these intranuclear inclusions and the other structures of the cellular nucleus are still obscure. The aim of this study is to elucidate the morphological and cytochemical peculiarities of intranuclear ubiquitin-immunopositive bodies in the substantia nigra neurons of human brain and to evaluate the interconnections of these peculiarities with nucleolus by means of light microscopy, immunocytochemistry, and confocal laser microscopy. It is found that up to 20% of neurons in substantia nigra of human brain contain ubiquitin-immunopositive Marinesco bodies. These rounded structures are 1–8 μm—more often 2–4 μm—in diameter. Only one-third of them are tightly adjacent to the nucleolus. By a method of silver impregnation of argentophilic proteins associated with nucleolar organizer, the absence was shown of argentophilic proteins, which are characteristic for the nucleolus, in Marinesco bodies. Special ubiquitin-positive substantially smaller structures (less than 1 μm) are revealed in the neurons’ nuclei along with Marinesco bodies. These structures are probably the initial forms in the formation of Marinesco bodies. The existence of two types of ubiquitin-immunopositive intranuclear bodies is revealed by means of confocal microscopy: one has high intensity of immunofluorescence, and the other has low intensity. Heterogeneous distribution of immunopositive product is characteristic of the former. The presence of DNA in Marinesco bodies is detected by using SYTOX Green fluorescent dye. The absence of peripheral heterochromatin zone and weak susceptibility to toluidine blue together with the presence of DNA and the absence of argentophilic proteins suggests substantial structural and chemical differences between Marinesco bodies and nucleoli, which argues against the idea that the detected bodies are modified nucleoli.

Iron, copper and their proteins in substantia nigra of human brain during aging

Scarce information is available on the content of metals and their molecules in the human brain. Iron, copper and other metals are involved in neurodegenerative disorders like Parkinson’s Disease (PD), however, their behavior in physiological conditions is poorly understood. In this study we have measured iron, copper and their major proteins (ferritins and ceruloplasmin) in substantia nigra (SN) of normal subjects at different ages, since this is the main target region of PD. An increasing trend for iron and copper concentration was found in aging. Ferritins were also increasing in aging while ceruloplasmin did not vary. These data show that the accumulation of these metals requires an increased expression of storage molecules to prevent toxic effects of iron and copper.

Combined biochemical separation and INAA for the determination of iron and other metals in Neuromelanin of human brain Substantia Nigra

In Parkinson disease Fe and other metals increase in Substantia Nigra(SN) and other basal nuclei. Since Fe can generate citotoxic free radicals,Neuromelanin (NM) could play an important protective role in neurons. In thiswork an original procedure for separation of NM, preparation of samples andanalysis is presented. The determination of SN and its NM elemental contentwas carried out by instrumental neutron activation analysis. Several actionswere taken to reduce the metal contaminations: use of high purity reagents,dissection of tissues with titanium coated tools and adequate processing ofsamples.

Enzymatic dopamine peroxidation in substantia nigra of human brain

The main metabolic pathway affected in Parkinson’s disease is that of dopamine oxidation and melanin formation in substantia nigra which involves both oxidative and reductive enzymes. The cyclic nature of the biosynthetic pathway from dopamine to melanin implies that a derangement at any of the steps may result in the disappearance of melanin. Possible pathogenetic events such as oxidative stress have therefore no clearcut interpretation since they may be both cause or consequence of the disease. This paper documents the existence of a peroxidase converting dopamine to dopaminochrome in the presence of hydrogen peroxide in the substantia nigra of autopsied human brain. The activatory effect of dopaminochrome on a purified peroxidase is shown, together with the inhibitory effect of dopaminochrome-derived melanin and the activatory effect of melanin/Fe. The toxic effect of dopaminochrome on murine neuroblastoma cells cultured in vitro is demonstrated together with the inhibition of dopaminochrome melanization induced by acetylcholine in vitro.

Cell death of dopamine neurons in aging and Parkinson’s disease

Dopamine neurons in the substantia nigra of human brain are selectively vulnerable and the number decline by aging at 5–10% per decade. Enzymatic and non-enzymatic oxidation of dopamine generates reactive oxygen species, which induces apoptotic cell death in dopamine neurons. Parkinson’s disease (PD) is also caused by selective cell death of dopamine neurons in this brain region. The pathogenesis of Parkinson’s disease remains to be an enigma, but it was found that an endogenous MPTP-like neurotoxin, 1( R), 2( N)-dimethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline [ N-methyl( R)salsolinol, NM( R)Sal], may be one of the pathogenic agents of PD. NM(R)Sal increases in cerebrospinal fluid from untreated parkinsonian patients, and two enzymes, a ( R)salsolinol synthase and a neutral N-methyltransferase, synthesize this neurotoxin in the nigro-striatum. The activity of a neutral N-methyltransferase is significantly higher in lymphocytes from parkinsonian patients than in control. The mechanism of cell death by this toxin was proved to be by the induction of apoptosis, by use of dopaminergic SH-SY5Y cells. The apoptosis was suppressed by anti-oxidants, suggesting that the generation of reactive oxygen species may initiate cellular death process. These results indicate that in aging and PD oxidative stress induces degeneration of dopamine neurons, and the antioxidant therapy may delay the decline of dopamine neurons in the brain.

Glutathione transferases catalyse the detoxication of oxidized metabolites (o-quinones) of catecholamines and may serve as an antioxidant system preventing degenerative cellular processes.

o-Quinones are physiological oxidation products of catecholamines that contribute to redox cycling, toxicity and apoptosis, i.e. the neurodegenerative processes underlying Parkinson's disease and schizophrenia. The present study shows that the cyclized o-quinones aminochrome, dopachrome, adrenochrome and noradrenochrome, derived from dopamine, dopa, adrenaline and noradrenaline respectively, are efficiently conjugated with glutathione in the presence of human glutathione transferase (GST) M2-2. The oxidation product of adrenaline, adrenochrome, is less active as a substrate for GST M2-2, and more efficiently conjugated by GST M1-1. Evidence for expression of GST M2-2 in substantia nigra of human brain was obtained by identification of the corresponding PCR product in a cDNA library. Glutathione conjugation of these quinones is a detoxication reaction that prevents redox cycling, thus indicating that GSTs have a cytoprotective role involving elimination of reactive chemical species originating from the oxidative metabolism of catecholamines. In particular, GST M2-2 has the capacity to provide protection relevant to the prevention of neurodegenerative diseases.

The effect of a synthetic neuromelanin on yield of free hydroxyl radicals generated in model systems

Neuromelanin is an amorphous pigment of the catecholamine origin that accumulates in certain dopaminergic neurons of the substantia nigra of human brain. In Parkinson's disease, there appears to be selective degeneration of the most heavily pigmented neurons of the substantia nigra, and this process has been linked to the presence of neuromelanin. It has been postulated that neuromelanin could increase the risk of oxidative stress reactions. On the other hand, melanin is usually considered to be an efficient antioxidant. Here we analyze experimental conditions that stimulate, or inhibit, antioxidant properties of neuromelanin. Using electron spin resonance (ESR) - spin trapping technique and salicylate hydroxylation assay, we monitored the formation of free hydroxyl radicals generated by a Fenton system in the presence of varying concentration of dopamine-melanin, a synthetic model for neuromelanin. Our data clearly indicate that the antioxidant action of neuromelanin is predominantly due to its ability to sequester redox-active metal ions such as iron. Using direct ESR spectroscopy, we have shown that ferric complexes with neuromelanin are resistant to reduction by mild biological reductants such as ascorbate. We have demonstrated that dopamine-melanin saturated with ferric ions, could enhance the formation of free hydroxyl radicals by redox activation of the ions. Thus, under the conditions that stimulate the release of accumulated metal ions, neuromelanin may actually become an efficient prooxidant. It is conceivable that neuromelanin, which normally is able to protect pigmented dopaminergic neurons against metal-ion related toxicity, could under extreme conditions have a cytotoxic role.

Potential bioactivated neurotoxicants, N-methylated β-carbolinium ions, are present in human brain

Potential bioactivated neurotoxicants, 2- N-methyl-β-carbolinium and 2,9- N,N′-dimethyl-β-carbolinium ions, as well as N-methylation activities which form these charged species, were analyzed for the first time in the parietal association cortex and the substantia nigra of human brain using GC/MS and HPLC. The brains were taken during forensic autopsies from corpses without obvious degeneration of substantia nigra. In the cortex, 2-methyl-norhamanium ion (2-MeNH) and 2,9-dimethyl-norharmanium ion (2,9-Me 2NH) were detected in almost alll samples. 2-Methyl-harmanium ions (2-MeHA) and 2,9-dimethyl-harmanium ions (2,9-Me 2HA) were detectable in only two samples. In substantia nigra samples pooled from 3 or 4 brains for analysis, 2-MeNH and 2,9-Me 2NH levels were higher than those in the cortex, whereas 2-MeHA and 2,9-Me 2HA were below detection limits. Their precursors, norharman (NH) and harman (HA), were also measured using HPLC/fluorescence detection. In both regions, NH and HA were present in almost all samples; levels of NH and HA were also significantly higher in the nigra than in the cortex. Using 9-methyl-NH and 2-MeNH as substrates, in vitro N-methylation of the 2[β] and 9[indole] nitrogens toward β-carbolines was measured both in the cortex and in the nigra. 2[β]- N-Methylation activity was significantly higher than 9[indole]- N-methylation activity in both regions. Recent studies show that β-carbolinium ions resemble the synthetic parkinsonian toxicant, MPP +, with respect to structure and neurotoxic activity. Such ‘bioactivated’ carbolinium ions could be endogenous causative factors in Parkinson's disease.

The chemical characterization of melanin contained in substantia nigra of human brain

The pigment of substantia nigra human brain has been extracted by a mild procedure consisting of washes with phosphate buffer, methanol and incubation with SDS-proteinase. Pyrolisis gas chromatography mass spectrometry infrared spectrometry, termogravimetric analysis and elemental analysis were the techniques used for the chemical characterization. An indole moiety bound to a sulfur containing amino acid and to palmitic acid were the main aspects found in the structure. The presence of a 7% inorganic component was observed. This probably contains Fe, Cu, Zn and Cr which are also relevant, for the formation and the role of melanin in substantia nigra neurons. The fatty acid moiety is chemically bound to the indole structure as it was not eliminated by repeated methanol washing. The same situation occurs for the sulfur containing gropu. Considering these data and the most abundant molecules present in substantia nigra the precursor of neuromelanin seems to be a cysteinyl-cethecol, to which is then bound a palmityl group.

Expression of a Human Cocaine‐Sensitive Dopamine Transporter in Xenopus laevis Oocytes

The human dopamine transporter was expressed in Xenopus laevis oocytes following injection of mRNA isolated from human brain substantia nigra. The specific accumulation of [3H]dopamine into these oocytes was time and Na+ dependent. Furthermore, [3H]dopamine accumulation was prevented by coincubation of oocytes with dopamine (100 microM) or with the dopamine uptake inhibitors GBR 12909 (1 microM) or cocaine (3 microM). In contrast, oocyte injection of mRNA isolated from human globus pallidus, an area devoid of dopamine neuron perikarya, did not elicit expression of the dopamine transporter. Oocyte expression of the human dopamine transporter can be used for the further characterization and cloning of this low-abundance membrane protein.

Identification of substance P precursor forms in human brain tissue.

Substance P prohormones were identified in the caudate nucleus, hypothalamus, and substantia nigra of human brain. A polypeptide fraction of acidic brain extracts was fractionated on Sephadex G-50. The lyophilized fractions were sequentially treated with trypsin and a substance P-degrading enzyme with strong preference toward the Phe7-Phe8 and Phe8-Gly9 bonds. The released substance P(1-7) fragment was isolated by ion-exchange chromatography and quantitated by a specific radioimmunoassay. Confirmation of the structure of the isolated radioimmunoassay-active fragment was achieved by electrophoresis and HPLC. By using this enzymatic/radioimmunoassay procedure, two polypeptide fractions of apparent Mr 5000 and 15,000, respectively, were identified. The latter component was the major one of the two but was estimated to account for only about 5% of total substance P radioimmunoassay activity. Because it is of the size predicted from the nucleotide sequences of cDNA for substance P prohormones in bovine brain, the Mr 15,000 component may represent the full-length prohormone.

Neuronal pigments: spectroscopic characterization of human brain melanin.

Abstract— A method has been established for the characterization of synthetic melanins obtained from dopamine, norepinephrine, and structurally related compounds which permits the identification of the precursor substrate of the melanin. This is done by interpreting the absorption spectra and the first derivative curves of these melanins solubilized in sodium borohydride. Using this method, the melanin pigment obtained from the substantia nigra of human brain has been shown to be very similar to dopamine‐melanin and not DOPA‐ or norepinephrine‐melanins.