Normal Human Substantia Nigra Research Articles

Elemental mapping of Neuromelanin organelles of human Substantia Nigra: correlative ultrastructural and chemical analysis by analytical transmission electron microscopy and nano-secondary ion mass spectrometry.

Neuromelanin (NM) is a compound which highly accumulates mainly in catecholamine neurons of the substantia nigra (SN), and is contained in organelles (NM-containing organelles) with lipid bodies and proteins. These neurons selectively degenerate in Parkinson's disease and NM can play either a protective or toxic role. NM-containing organelles of SN were investigated by Analytical Electron Microscopy (AEM) and Nano-Secondary Ion Mass Spectrometry (NanoSIMS) within human tissue sections with respect to ultrastructure and elemental composition. Within the NM-containing organelle, the single NM granules and lipid bodies had sizes of about 200-600nm. Energy-Dispersive X-ray microanalysis spectra of the NM granules and lipid bodies were acquired with 100nm beam diameter in AEM, NanoSIMS yielded elemental maps with a lateral resolution of about 150nm. AEM yielded the quantitative elemental composition of NM granules and bound metals, e.g., iron with a mole fraction of about 0.15 atomic percent. Chemical analyses by AEM and NanoSIMS were consistent at the subcellular level so that nanoSIMS measurements have been quantitated. In NM granules of SN from healthy subjects, a significant amount of S, Fe, and Cu was found. In lipid bodies an amount of P consistent with the presence of phospholipids was measured. The improved detection limits of nanoSIMS offer new possibilities for chemical mapping, high-sensitivity trace element detection, and reduced acquisition times. Variations between individual NM granules can now be investigated effectively and quantitatively by NanoSIMS mapping Cu and Fe. This should yield new insight into the changes in chemical composition of NM pigments during healthy aging and disease. Neuromelanin-containing organelles of dopamine neurons in normal human substantia nigra were investigated by analytical electron mircoscopy and secondary ion mass spectroscopy (NanoSIMS) yielding the ultrastructure and elemental composition. In neuromelanin granules a significant amount of S, Fe and Cu was found. In lipid bodies an amount of P consistent with the presence of phospholipids was measured. The improved sensitivity of NanoSIMS shows differences in chemical composition between individual neuromelanin granules and allows to study chemical changes of neuromelanin organelles during aging and disease.

Normal substantia nigra patients treated with levodopa - Clinical, therapeutic and pathological observations.

Definite diagnosis of idiopathic Parkinson's disease is based on histological findings of marked substantia nigra neuronal loss and Lewy body inclusions. Almost all cases with clinical diagnosis of idiopathic Parkinson's disease are treated with levodopa. Because there is no biological marker for the diagnosis, erroneous clinical diagnosis and treatment of such cases with levodopa are well known. There is very limited literature on levodopa treated cases that had normal substantia nigra at autopsy. Patients seen at Movement Disorders Clinic Saskatchewan are offered autopsy at no cost to the family/estate of the patient. Autopsy studies are performed by certified neuropathologists. Notation on the status of substantia nigra is made in every autopsied case. Between 1968 and 2014, 21 cases treated with levodopa had normal substantia nigra at autopsy. Eleven patients continued levodopa until death and 9 received the drug for four years or longer. No objective motor symptom benefit, dyskinesia or motor response fluctuations on levodopa were observed in any case. The most common final diagnosis was essential tremor. Individuals with normal substantia nigra do not benefit from levodopa and do not manifest motor response fluctuations or dyskinesia. Long-term use of levodopa is not toxic to normal human substantia nigra.

Differentiation of Human Epidermal Neural Crest Stem Cells (hEPI-NCSC) into Virtually Homogenous Populations of Dopaminergic Neurons

Here we provide a protocol for the directed differentiation of hEPI-NCSC into midbrain dopaminergic neurons, which degenerate in Parkinson’s disease. hEPI-NCSC are neural crest-derived multipotent stem cells that persist into adulthood in the bulge of hair follicles. The experimental design is distinctly different from conventional protocols for embryonic stem cells and induced pluripotent stem (iPS) cells. It includes pre-differentiation of the multipotent hEPI-NCSC into neural stem cell-like cells, followed by ventralizing, patterning, continued exposure to the TGFβ receptor inhibitor, SB431542, and at later stages of differentiation the presence of the WNT inhibitor, IWP-4. All cells expressed A9 midbrain dopaminergic neuron progenitor markers with gene expression levels comparable to those in normal human substantia nigra. The current study shows for the first time that virtually homogeneous populations of dopaminergic neurons can be derived ex vivo from somatic stem cells without the need for purification, with useful timeliness and high efficacy. This novel development is an important first step towards the establishment of fully functional dopaminergic neurons from an ontologically relevant stem cell type, hEPI-NCSC.Electronic supplementary materialThe online version of this article (doi:10.1007/s12015-013-9493-9) contains supplementary material, which is available to authorized users.

Osteopontin expression in substantia nigra in MPTP-treated primates and in Parkinson's disease

Parkinson's disease (PD) is characterised by the loss of dopaminergic neurones in the substantia nigra (SN) but the pathogenic mechanism remains unknown. Cell death involves oxidative stress and inflammatory mechanisms, and these may be altered by the actions of the glycosylated phosphoprotein osteopontin (OPN). OPN is present in the rat SN, but its presence in human and non-human primate brain has not been extensively studied. Both OPN mRNA and protein were present in the normal marmoset SN, and OPN protein was localised to nigral neurones although these were not dopaminergic cells and it was not present in glial cells. In contrast, OPN protein was found in dopaminergic neurones in the normal human SN but again not in glial cells with some accumulation in the extracellular matrix. Following MPTP treatment of common marmosets, OPN protein expression was decreased, although its mRNA levels were unchanged and it was not present in either activated microglia or astrocytes. In the SN in PD, OPN protein expression was decreased in the remaining dopaminergic neurones and it was present in activated microglia but not in astrocytes. This was not specific to PD as OPN protein expression was also decreased in the SN in multiple system atrophy and progressive supranuclear palsy with an identical localisation of the protein. The presence of OPN in the normal human and non-human primate SN coupled to its decreased expression following nigral cell degeneration suggests that it may play an important role in dopaminergic neurone survival.

The Neuromelanin of Human Substantia Nigra: Physiological and Pathogenic Aspects

Neuromelanin (NM) accumulates as a function of age in normal human substantia nigra (SN) but is relatively depleted in the SN of patients with Parkinson disease (PD). Several studies have been performed to further our understanding of the role of NM in neuronal aging and neurodegenerative mechanisms of PD. To this purpose, NM from human SN was isolated and its structure and molecular interactions were investigated. Cysteinyl-dopamine was shown to be one precursor of NM synthesis. A striking affinity of NM for specific metals, lipids, drugs and pesticides was found in vitro, and in animal and human brain postmortem studies. Because of these affinities, NM seems to play a protective role in the human brain by blocking toxic molecules. On the other hand, experiments in cell culture indicate that NM can activate microglia, eliciting the release of cytotoxic factors that can induce neurodegeneration.

Alterations in m-RNA expression for Cu,Zn-superoxide dismutase and glutathione peroxidase in the basal ganglia of MPTP-treated marmosets and patients with Parkinson’s disease

Alterations occurring in the antioxidant enzymes, copper, zinc-dependent superoxide dismutase (Cu,Zn-SOD) and glutathione peroxidase (GPX) following nigral dopaminergic denervation are unclear. We now report on the distribution and levels of m-RNA for Cu,Zn-SOD and GPX in basal ganglia of normal and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated common marmosets, and in normal individuals and patients with Parkinson’s disease (PD) using in situ hybridization histochemistry and oligodeoxynucleotide (single-stranded DNA) probes. Cu,Zn-SOD and GPX m-RNA was present throughout basal ganglia (nucleus accumbens, caudate-putamen, globus pallidus, substantia nigra) in the common marmoset, with the highest levels being in substantia nigra (SN). Following MPTP induced nigral cell loss, Cu,Zn-SOD m-RNA levels were decreased in all areas but the SNr, and particularly in SNc (71%, P<0.001). MPTP-treatment had no effect on GPX m-RNA expression in any area of basal ganglia. Cu,Zn-SOD and GPX m-RNA was also present in the normal human SN. In PD, however, Cu,Zn-SOD m-RNA was significantly decreased (89%, P<0.005) in SNc, and there was a near-complete loss of GPX m-RNA in both SNc (100%, P<0.005) and SNr (88%, P<0.005). The loss of Cu,Zn-SOD m-RNA in SNc in MPTP-treated marmosets and patients with PD suggests that it is primarily located in dopaminergic neuronal cell bodies. The loss of GPX m-RNA in SNc in PD also suggests a localisation to dopaminergic cell bodies, but the similar change in SNr may indicate its presence in dopaminergic neurites. In contrast, the absence of change in GPX m-RNA in MPTP-treated primates appears to rule out its presence in dopaminergic cells in this species, but this may only be apparent and may reflect increased expression in glial cells following acute toxin treatment.

Lactoferrin is synthesized by activated microglia in the human substantia nigra and its synthesis by the human microglial CHME cell line is upregulated by tumor necrosis factor α or 1-methyl-4-phenylpyridinium treatment

The presence of the iron-binding protein lactoferrin (Lf) in some specific areas of the central nervous system and particularly in the normal human substantia nigra, where it is found in dopaminergic (DA) neurons and some glial cells, led us to investigate Lf synthesis in this area. Lf mRNA were identified using in situ hybridization and found in small ameboid cells. These cells were identified using immunocytochemistry as activated microglia since they exhibited macrophage markers such as the CD68 and the CR1 antigens. Double immunofluorescent labeling confirmed that the two Lf immunostained cell populations were activated microglia and DA neurons. Since activated microglia contained both Lf and its messenger, these cells are the Lf producing cells. The presence of Lf in DA neurons in which no Lf messengers were visible, might be due to an endocytosis mechanism, DA neurons probably internalizing Lf produced in microglial cells located in their neighborhood. In neuropathological disorders, such as Alzheimer’s and Parkinson’s diseases, inflammatory process and oxidative stress are events that contribute to neuronal death. Since Lf concentration increases during these pathologies, we studied the level of Lf expression under these different stresses and showed, using RT-PCR, that the immortalized human embryonic microglial CHME cell line produced Lf transcripts under tumor necrosis factor α or 1-methyl-4-phenylpyridinium treatment whereas untreated cells did not. These data confirm that Lf is produced only when microglia are activated.

Isolation and 13C-NMR characterization of an insoluble proteinaceous fraction from substantia nigra of patients with Parkinson's disease.

Neuromelanin is a dark brown pigment suspected of being involved in the pathogenesis of Parkinson's disease. This pigment can be isolated from normal human substantia nigra by a procedure that includes an extensive proteolytic treatment. In this study we used such a procedure to extract the neuromelanin pigment from a pool of substantia nigra from patients affected by Parkinson's disease. 13C Cross polarization magic angle spinning nuclear magnetic resonance spectroscopy and electron paramagnetic resonance spectroscopy were used to characterize the solid residue obtained from the extraction procedure. We found that the pigment extracted from the substantia nigra of parkinsonian patients was mainly composed of highly cross-linked, protease-resistant, lipo-proteic material, whereas the neuromelanin macromolecule appears to be only a minor component of this extract. A synthetic model of melanoprotein has been prepared by enzymatic oxidation of dopamine in the presence of albumin. Once it has undergone the same proteolytic treatment, this model system yields a 13C-NMR spectrum which is similar to that observed for the parkinsonian midbrain extract. These results are consistent with the view that oxidative stress has a relevant role in the pathogenesis of Parkinson's disease.

Purified human neuromelanin, synthetic dopamine melanin as a potential model pigment, and the normal human substantia nigra: characterization by electron paramagnetic resonance spectroscopy.

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.

Parkinson's disease: depletion of substantia nigra neurotensin receptors

Neurotensin receptor autoradiography reveals dense binding in normal human substantia nigra. In nigra from patients with Parkinson's disease, nigral receptor binding is only about one-third of control values. ‘Saturation’ analysis suggests that these changes result from receptor loss. These results support a neurotensin-domapine interaction in human nigro-striatal circuits.

Tyrosinase-like activity in normal human substantia nigra

1. 1. A tyrosinase-like activity was found in human substantia nigra by polyacrylamide gel electrophoresis of fractions prepared from homogenates of the substantia nigra. 2. 2. The enzyme activity was detected by staining the gels with l-3,4-dihydroxyphenylalanine, dopamine and 5,6-dihydroxyindole as substrates for tyrosinase (EC 1.14.18.1). 3. 3. A case of parkinsonism does not show the l-3,4-dihydroxyphenylalanine and dopamine oxidase activities.