Human Substantia Nigra Pars Compacta Research Articles

Parkinson's Disease: Cells Succumbing to Lifelong Dopamine-Related Oxidative Stress and Other Bioenergetic Challenges.

The core pathological event in Parkinson's disease (PD) is the specific dying of dopamine (DA) neurons of the substantia nigra pars compacta (SNc). The reasons why SNc DA neurons are especially vulnerable and why idiopathic PD has only been found in humans are still puzzling. The two main underlying factors of SNc DA neuron vulnerability appear related to high DA production, namely (i) the toxic effects of cytoplasmic DA metabolism and (ii) continuous cytosolic Ca2+ oscillations in the absence of the Ca2+-buffer protein calbindin. Both factors cause oxidative stress by producing highly reactive quinones and increasing intra-mitochondrial Ca2+ concentrations, respectively. High DA expression in human SNc DA neuron cell bodies is suggested by the abundant presence of the DA-derived pigment neuromelanin, which is not found in such abundance in other species and has been associated with toxicity at higher levels. The oxidative stress created by their DA production system, despite the fact that the SN does not use unusually high amounts of energy, explains why SNc DA neurons are sensitive to various genetic and environmental factors that create mitochondrial damage and thereby promote PD. Aging increases multiple risk factors for PD, and, to a large extent, PD is accelerated aging. To prevent PD neurodegeneration, possible approaches that are discussed here are (1) reducing cytoplasmic DA accumulation, (2) blocking cytoplasmic Ca2+ oscillations, and (3) providing bioenergetic support.

Neuromelanin accumulation drives endogenous synucleinopathy in non-human primates.

Although neuromelanin is a dark pigment characteristic of dopaminergic neurons in the human substantia nigra pars compacta, its potential role in the pathogenesis of Parkinson's disease (PD) has often been neglected since most commonly used laboratory animals lack neuromelanin. Here we took advantage of adeno-associated viral vectors encoding the human tyrosinase gene for triggering a time-dependent neuromelanin accumulation within substantia nigra pars compacta dopaminergic neurons in macaques up to similar levels of pigmentation as observed in elderly humans. Furthermore, neuromelanin accumulation induced an endogenous synucleinopathy mimicking intracellular inclusions typically observed in PD together with a progressive degeneration of neuromelanin-expressing dopaminergic neurons. Moreover, Lewy body-like intracellular inclusions were observed in cortical areas of the frontal lobe receiving dopaminergic innervation, supporting a circuit-specific anterograde spread of endogenous synucleinopathy by permissive trans-synaptic templating. In summary, the conducted strategy resulted in the development and characterization of a new macaque model of PD matching the known neuropathology of this disorder with unprecedented accuracy. Most importantly, evidence is provided showing that intracellular aggregation of endogenous α-synuclein is triggered by neuromelanin accumulation, therefore any therapeutic approach intended to decrease neuromelanin levels may provide appealing choices for the successful implementation of novel PD therapeutics.

The Proteome of Neuromelanin Granules in Dementia with Lewy Bodies.

Neuromelanin granules (NMGs) are organelle-like structures present in the human substantia nigra pars compacta. In addition to neuromelanin, NMGs contain proteins, lipids and metals. As NMG-containing dopaminergic neurons are preferentially lost in Parkinson's disease and dementia with Lewy bodies (DLB), it is assumed that NMGs may play a role in neurodegenerative processes. Until now, this role is not completely understood and needs further investigation. We therefore set up an exploratory proteomic study to identify differences in the proteomic profile of NMGs from DLB patients (n = 5) compared to healthy controls (CTRL, n = 5). We applied a laser microdissection and mass-spectrometry-based approach, in which we used targeted mass spectrometric experiments for validation. In NMG-surrounding (SNSurr.) tissue of DLB patients, we found evidence for ongoing oxidative damage and an impairment of protein degradation. As a potentially disease-related mechanism, we found α-synuclein and protein S100A9 to be enriched in NMGs of DLB cases, while the abundance of several ribosomal proteins was significantly decreased. As S100A9 is known to be able to enhance the formation of toxic α-synuclein fibrils, this finding points towards an involvement of NMGs in pathogenesis, however the exact role of NMGs as either neuroprotective or neurotoxic needs to be further investigated. Nevertheless, our study provides evidence for an impairment of protein degradation, ongoing oxidative damage and accumulation of potentially neurotoxic protein aggregates to be central mechanisms of neurodegeneration in DLB.

Early Forms of α-Synuclein Pathology Are Associated with Neuronal Complex I Deficiency in the Substantia Nigra of Individuals with Parkinson's Disease.

Idiopathic Parkinson’s disease (iPD) is characterized by degeneration of the dopaminergic substantia nigra pars compacta (SNc), typically in the presence of Lewy pathology (LP) and mitochondrial respiratory complex I (CI) deficiency. LP is driven by α-synuclein aggregation, morphologically evolving from early punctate inclusions to Lewy bodies (LBs). The relationship between α-synuclein aggregation and CI deficiency in iPD is poorly understood. While studies in models suggest they are causally linked, observations in human SNc show that LBs preferentially occur in CI intact neurons. Since LBs are end-results of α-synuclein aggregation, we hypothesized that the relationship between LP and CI deficiency may be better reflected in neurons with early-stage α-synuclein pathology. Using quadruple immunofluorescence in SNc tissue from eight iPD subjects, we assessed the relationship between neuronal CI or CIV deficiency and early or late forms of LP. In agreement with previous findings, we did not observe CI-negative neurons with late LP. In contrast, early LP showed a significant predilection for CI-negative neurons (p = 6.3 × 10−5). CIV deficiency was not associated with LP. Our findings indicate that early α-syn aggregation is associated with CI deficiency in iPD, and suggest a double-hit mechanism, where neurons exhibiting both these pathologies are selectively lost.

Evidence That Substantia Nigra Pars Compacta Dopaminergic Neurons Are Selectively Vulnerable to Oxidative Stress Because They Are Highly Metabolically Active.

There are 400–500 thousand dopaminergic cells within each side of the human substantia nigra pars compacta (SNpc) making them a minuscule portion of total brain mass. These tiny clusters of cells have an outsized impact on motor output and behavior as seen in disorders such as Parkinson’s disease (PD). SNpc dopaminergic neurons are more vulnerable to oxidative stress compared to other brain cell types, but the reasons for this are not precisely known. Here we provide evidence to support the hypothesis that this selective vulnerability is because SNpc neurons sustain high metabolic rates compared to other neurons. A higher baseline requirement for ATP production may lead to a selective vulnerability to impairments in oxidative phosphorylation (OXPHOS) or genetic insults that impair Complex I of the electron transport chain. We suggest that the energy demands of the unique morphological and electrophysiological properties of SNpc neurons may be one reason these cells produce more ATP than other cells. We further provide evidence to support the hypothesis that transcription factors (TFs) required to drive induction, differentiation, and maintenance of midbrain dopaminergic neural progenitor cells which give rise to terminally differentiated SNpc neurons are uniquely involved in both developmental patterning and metabolism, a dual function unlike other TFs that program neurons in other brain regions. The use of these TFs during induction and differentiation may program ventral midbrain progenitor cells metabolically to higher ATP levels, allowing for the development of those specialized cell processes seen in terminally differentiated cells. This paper provides a cellular and developmental framework for understanding the selective vulnerability of SNpc dopaminergic cells to oxidative stress.

Co-Expression of Nogo-A in Dopaminergic Neurons of the Human Substantia Nigra Pars Compacta Is Reduced in Parkinson's Disease.

Parkinson’s disease is mainly characterized by a progressive loss of dopaminergic neurons in the substantia nigra pars compacta. Together with the small number, the high vulnerability of the dopaminergic neurons is a major pathogenic culprit of Parkinson’s disease. Our previous findings of a higher survival of dopaminergic neurons in the substantia nigra co-expressing Nogo-A in an animal model of Parkinson’s disease suggested that Nogo-A may be associated with dopaminergic neurons resilience against Parkinson’s disease neurodegeneration. In the present study, we have addressed the expression of Nogo-A in the dopaminergic neurons in the substantia nigra in postmortem specimens of diseased and non-diseased subjects of different ages. For this purpose, in a collaborative effort we developed a tissue micro array (TMA) that allows for simultaneous staining of many samples in a single run. Interestingly, and in contrast to the observations gathered during normal aging and in the animal model of Parkinson’s disease, increasing age was significantly associated with a lower co-expression of Nogo-A in nigral dopaminergic neurons of patients with Parkinson’s disease. In sum, while Nogo-A expression in dopaminergic neurons is higher with increasing age, the opposite is the case in Parkinson’s disease. These observations suggest that Nogo-A might play a substantial role in the vulnerability of dopaminergic neurons in Parkinson’s disease.

Spatial RNA Sequencing Identifies Robust Markers of Vulnerable and Resistant Human Midbrain Dopamine Neurons and Their Expression in Parkinson's Disease.

Defining transcriptional profiles of substantia nigra pars compacta (SNc) and ventral tegmental area (VTA) dopamine neurons is critical to understanding their differential vulnerability in Parkinson’s Disease (PD). Here, we determine transcriptomes of human SNc and VTA dopamine neurons using LCM-seq on a large sample cohort. We apply a bootstrapping strategy as sample input to DESeq2 and identify 33 stably differentially expressed genes (DEGs) between these two subpopulations. We also compute a minimal sample size for identification of stable DEGs, which highlights why previous reported profiles from small sample sizes display extensive variability. Network analysis reveal gene interactions unique to each subpopulation and highlight differences in regulation of mitochondrial stability, apoptosis, neuronal survival, cytoskeleton regulation, extracellular matrix modulation as well as synapse integrity, which could explain the relative resilience of VTA dopamine neurons. Analysis of PD tissues showed that while identified stable DEGs can distinguish the subpopulations also in disease, the SNc markers SLIT1 and ATP2A3 were down-regulated and thus appears to be biomarkers of disease. In summary, our study identifies human SNc and VTA marker profiles, which will be instrumental for studies aiming to modulate dopamine neuron resilience and to validate cell identity of stem cell-derived dopamine neurons.

CD8 T cell nigral infiltration precedes synucleinopathy in early stages of Parkinson's disease.

There is no consensus on the exact role of the adaptive immune system in Parkinson's disease pathogenesis, although there is increasing evidence that it is somehow involved. Moreover, T cell infiltration in the brain has not been thoroughly studied in Parkinson's disease and no study has assessed the infiltration in incidental Lewy body diseases cases that are considered to be early presymptomatic stages of the disease. In this study, we performed an immunohistochemistry/immunofluorescence quantitative and phenotypic assessment of T cell infiltration in human substantia nigra pars compacta and analysed the correlations with neuronal death and synucleinopathy throughout different stages of the disease. We included two groups of incidental Lewy disease in the study. One of the groups, which is believed to be the earliest stage of the disease, showed α-synuclein aggregates only in the olfactory bulb. The second group also presented α-synuclein aggregates in the substantia nigra. We also assessed the formation of different α-synuclein aggregates throughout the different stages of the unified staging system for Lewy body disorders (I to IV). We found that CD8 T cells were increased in diagnosed Parkinson's disease cases compared to the control group and their density positively correlated with neuronal death. Some of the infiltrating CD8 T cells were indeed contacting dopaminergic neurons. No differences were found regarding CD4 T cells. In the earliest stage of the disease, when substantia nigra α-synuclein aggregation is absent, we found a robust CD8 T cell infiltration and no dopaminergic neuronal death yet. Conversely, in the next stage we found neuronal loss and a milder CD8 T cell infiltration. CD8 T cell infiltration paralleled that of α-synuclein accumulation and neuronal death throughout stages II to IV. We also confirmed that CD8 T cells in charge of immune surveillance and involved in the aetiopathogenesis of the disease are equipped with cytolytic enzymes (granzyme A, B and K) and/or proinflammatory cytokines (interferon gamma), and that phenotypic differences were observed between early and late stages of the disease. We also demonstrate that a high proportion of nigral CD8 T cells are tissue resident memory T cells. Our results show that nigral cytotoxic CD8 T cell infiltration is an earlier pathogenic event than α-synuclein aggregation and neuronal death and that it parallels the progression of neuronal death and synucleinopathy in Parkinson's disease. Overall, our study suggests that CD8 T cell cytotoxic attack may initiate and propagate neuronal death and synucleinopathy in Parkinson's disease.

Age-dependent nigral dopaminergic neurodegeneration and α-synuclein accumulation in RGS6-deficient mice.

Parkinson's is primarily a non-familial, age-related disorder caused by α-synuclein accumulation and the progressive loss of dopamine neurons in the substantia nigra pars compacta (SNc). G protein-coupled receptor (GPCR)-cAMP signaling has been linked to a reduction in human Parkinson's incidence and α-synuclein expression. Neuronal cAMP levels are controlled by GPCRs coupled to Gs or Gi/o, which increase or decrease cAMP, respectively. Regulator of G protein signaling 6 (RGS6) powerfully inhibits Gi/o signaling. Therefore, we hypothesized that RGS6 suppresses D2 autoreceptor- Gi/o signaling in SNc dopamine neurons promoting neuronal survival and reducing α-synuclein expression. Here we provide novel evidence that RGS6 critically suppresses late-age-onset SNc dopamine neuron loss and α-synuclein accumulation. RGS6 is restrictively expressed in human SNc dopamine neurons and, despite their loss in Parkinson's, all surviving neurons express RGS6. RGS6-/- mice exhibit hyperactive D2 autoreceptors with reduced cAMP signaling in SNc dopamine neurons. Importantly, RGS6-/- mice recapitulate key sporadic Parkinson's hallmarks, including: SNc dopamine neuron loss, reduced nigrostriatal dopamine, motor deficits, and α-synuclein accumulation. To our knowledge, Rgs6 is the only gene whose loss phenocopies these features of human Parkinson's. Therefore, RGS6 is a key regulator of D2R-Gi/o signaling in SNc dopamine neurons, protecting against Parkinson's neurodegeneration and α-synuclein accumulation.

Regulator of G protein Signaling 6 (RGS6) Suppresses Late‐age‐onset Nigral Dopaminergic Neurodegeneration, α‐Synuclein Accumulation and Motor Dysfunction

Parkinson's disease (PD) is devastating, primarily non‐familial, age‐related neurodegenerative disorder caused by the progressive loss of dopamine (DA) neurons in the substantia nigra pars compacta (SNc). Why these neurons die with aging in sporadic PD is unknown. However, accumulation of α‐synuclein aggregates in Lewy bodies is believed to play a crucial role in PD pathogenesis. In support of this hypothesis, cAMP‐elevating β‐agonists dramatically reduce both α‐synuclein expression and the incidence of human PD as well as repress MPTP‐induced SNc DA neuron loss in mice (Mittal et al., Science 2017). Neuronal cAMP levels are controlled by neurotransmitter GPCRs coupled to Gs or Gi/o which stimulate or inhibit adenylyl cyclases (AC), respectively. RGS proteins powerfully modulate signaling by GPCRs including DA receptors. Remarkably, we have recently discovered that RGS6 is restrictively expressed in human SNc DA neurons that are lost in PD and knockout of RGS6 in mice recapitulates key hallmarks of sporadic PD, including: late‐age‐onset SNc DA neuron degeneration, reduced nigrostriatal DA, and motor deficits. We also demonstrated that RGS6 functions as a critical negative regulator of SNc D2 autoreceptor signaling allowing for proper modulation of DA‐dependent motor behaviors. Given that β‐agonists repress α‐synuclein expression through their activation of β2‐adrenorecetor‐Gαs, a GPCR that increases cAMP, and RGS6's role in inhibiting D2 autoreceptor signaling, a Gi/o GPCR that reduces cAMP, we hypothesize that RGS6, like β‐agonists, may function to prevent the pathological accumulation of α‐synuclein through modulation of cAMP‐PKA signaling. Here we show that RGS6 is a key negative modulator of Gi/o signaling in SNc DA neurons, with its loss provoking enhance D2 autoreceptor activity and a subsequent reduction in cAMP‐PKA signaling. These data are important as cAMP‐PKA signaling has not only been linked to SNc DA neuron survival in PD but also pathological α‐synuclein accumulation. In agreement with these previous reports, we observed elevated SNc α‐synuclein protein expression in 12 and 18mo RGS6−/− mice relative to RGS6+/+ controls. The aberrant elevation in α‐synuclein expression in the SNc of RGS6−/− mice was accompanied by a transition in α‐synuclein expression patterns from largely intracellular, as seen in RGS6+/+ mice, to largely extracellular. Furthermore, western blot analysis revealed increased expression of oligomeric α‐synuclein isoforms in the SNc of RGS6−/− mice, another hallmark of PD pathophysiology. Together, these data identify RGS6 as a key G protein regulator that protects against PD‐related neurodegeneration and α‐synuclein pathology through inhibition of D2R‐Gi/o signaling in SNc DA neurons.Support or Funding InformationNIH AA025919 and Interdisciplinary Research Fellowship T32HL007121This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Regulator of G‐protein signaling 6 (RGS6) expression in human substantia nigra pars compacta (SNc) and loss in Parkinson's disease (PD)

Parkinson's disease (PD) is a devastating neurodegenerative disorder characterized by the progressive loss of dopamine (DA) neurons in the SNc. The reason for the greater sensitivity of these neurons to degeneration and their loss with aging in PD is unknown. Although there are treatments targeting symptoms of this disease there are no interventions that protect DA neurons from degeneration. We and our collaborators have recently discovered that RGS6 is restrictively expressed in and required for the adult survival of SNc DA neurons that undergo degeneration in PD. Mice lacking RGS6 exhibit a late onset age‐dependent loss of SNc DA neurons accompanied by reduced striatal and SNc DA content and motor deficits as well as increased sensitivity to 6‐OHDA‐induced neurodegeneration and behavioral deficits. These findings suggest a critical pathogenic link between RGS6 loss and DA neuron degeneration in PD. Here we tested this hypothesis by a quantitative analysis of RGS6 expressing neurons in the SNc of human control and PD patients (n = 5 each). Immunohistochemical analysis of RGS6 and tyrosine hydroxylase (TH) expression and unbiased sterology was used to quantify the numbers of RGS6+ and TH+ SNc neurons. We found that RGS6 is exclusively expressed in DA neurons in the SNc of humans and there is a dramatic (73%) and selective loss of these RGS6+ TH+ neurons in PD patients. Consistent with our hypothesis, all surviving SNc TH+ neurons in normal and PD patients express RGS6. Given our evidence that RGS6 is required for survival of SNc DA neurons in mice, these findings suggest that RGS6 loss in human SNc DA neurons renders them susceptible to neurodegeneration characteristic of PD. These findings provide new evidence suggesting that RGS6 may have a critical neuroprotective role in PD in both mice and humans. Together our findings identify RGS6 as a potential novel therapeutic target for PD treatment.Support or Funding InformationNIH CA161882, MJ Fox 11551

Characterization of a Novel Monoclonal Antibody against Human Mitochondrial Ferritin and Its Immunohistochemical Application in Human and Monkey Substantia Nigra.

Mitochondrial ferritin (FtMt) is a novel iron storage protein with high homology to H-ferritin. Unlike the ubiquitously expressed H- and L-ferritin, FtMt is expressed in specific tissues such as the testis, heart, and brain. The function of FtMt is not fully understood; however, evidence suggests that it has a neuroprotective role in neurodegenerative diseases. We have previously reported that FtMt is expressed in catecholaminergic neurons of the monkey brainstem. To explore FtMt expression in human dopaminergic neurons, we designed a novel monoclonal antibody, C65-2, directed against human FtMt. Here, we report the properties of our C65-2 antibody. Western blots analysis and immunoabsorption tests demonstrated that the C65-2 antibody specifically recognized FtMt with no cross-reactivity to H-ferritin. Immunohistochemistry showed that the C65-2 antibody detected FtMt in neurons of the substantia nigra pars compacta (SNc) in humans and monkeys. We confirmed that FtMt is expressed in dopaminergic neurons of the human SNc. Our results suggest that FtMt is involved in various physiological and pathological mechanisms in human dopaminergic neurons, and the C65-2 monoclonal antibody promises to be a useful tool for determining the localization and biological functions of FtMt in the brain.

Aging causes morphological alterations in astrocytes and microglia in human substantia nigra pars compacta

Age being a risk factor for Parkinson's disease, assessment of age-related changes in the human substantia nigra may elucidate its pathogenesis. Increase in Marinesco bodies, α-synuclein, free radicals and so forth in the aging nigral neurons are clear indicators of neurodegeneration. Here, we report the glial responses in aging human nigra. The glial numbers were determined on Nissl-stained sections. The expression of glial fibrillary acidic protein, S100β, 2′, 3′-cyclic nucleotide 3′ phosphodiesterase, and Iba1 was assessed on cryosections of autopsied midbrains by immunohistochemistry and densitometry. The glial counts showed a biphasic increase, of which, the first prominent phase from fetal age to birth could be physiological gliogenesis whereas the second one after middle age may reflect mild age-related gliosis. Astrocytic morphology was altered, but glial fibrillary acidic protein expression increased only mildly. Presence of type-4 microglia suggests possibility of neuroinflammation. Mild reduction in 2′, 3′-cyclic nucleotide 3′ phosphodiesterase–labeled area denotes subtle demyelination. Stable age-related S100β expression indicates absence of calcium overload. Against the expected prominent gliosis, subtle age-related morphological alterations in human nigral glia attribute them a participatory role in aging.

Synchrotron radiation based X-ray fluorescence shows changes in the elemental composition of the human substantia nigra in aged brains.

Human brain aging is considered to be the leading risk factor for a variety of neurodegenerative alterations. In particular, it is thought that the human substantia nigra might play a pivotal role in age-associated dopamine depletion which could be responsible for neuronal demise and subsequent emergence of different neurological alterations. A plethora of neurochemical redox- and non-redox-driven mechanisms is mainly associated with modifications in the elemental composition of both neuromelanin-pigmented neurons and extraneuronal spaces in the human substantia nigra pars compacta (SNpc). An age-associated variation in the content of Fe, Cu, Zn and Ca has recently received great interest in neurology, as these elements are implicated in different biochemical mechanisms underlying malicious neurodegenerative disorders, including Parkinson's and Alzheimer's diseases. Interestingly, to the best our knowledge, there is lack of a comprehensive study on age-associated variation in the elemental composition of the human SNpc. In that respect, the aim of the present study was to make a preliminary attempt to unravel some of the age-associated mechanisms responsible for the metabolism of some redox-active and redox-inactive elements in the elderly. To do so, substantia nigra tissue specimens, drawn from 37 individuals who deceased without any signs of neurodegeneration, were subjected to spectroscopic studies using synchrotron radiation based X-ray fluorescence. Both neuromelanin-pigmented neurons and extraneuronal areas were studied. It appears that in the neurons, Fe tends to decrease, whilst Cu, Zn and Ca were found to accumulate as an individual gets older.

Targeting Skp1, an Ubiquitin E3 Ligase Component Found Decreased in Sporadic Parkinson’s Disease

Microarray-derived transcriptomic studies in human substantia nigra pars compacta (SNpc) samples from sporadic Parkinson’s disease (SPD) cases have opened an avenue to concentrate on potential gene intersections or cross-talks along the dopaminergic (DAergic) neurodegenerative cascade in SPD. One emerging gene candidate identified by our group was SKP1A (p19, S-phase kinase-associated protein 1A), found significantly decreased in the SNpc. It is part of the SCF (Skp1, Cullin 1, F-box protein) complex, the largest class of sophisticated ubiquitin-proteasome/E3 ligases, and can directly interact with Fbxo7, a gene defective in PARK15-linked PD. In vitro target validation by viral-mediated RNA interference revealed that the deficiency of Skp1 in a mouse SN-derived DAergic neuronal cell line potentiated the damage caused by exogenous insults implicated in PD pathology and caused the death of neurons undergoing differentiation, which developed Lewy body-like, α-synuclein-positive inclusions preceding cell death. Furthermore, recent animal studies show that site-directed intranigral stereotaxic injections of lentiviruses targeting SKP1A induce pathological and behavioral deficits in mice, supporting a significant role of Skp1 in SN DAergic neuronal survival in SPD. Thus, strategies aimed at increasing the activity or content of Skp1 may represent a novel therapeutic approach that has the potential to treat PD.

Neuronal vulnerability in Parkinson's disease

The classic motor symptoms of Parkinson's disease result from the progressive death of dopaminergic neurons within the substantia nigra. To date the relatively selective vulnerability of this brain region is not understood. The unique feature of dopaminergic neurons of the human substantia nigra pars compacta is the presence of the polymer pigment neuromelanin which gives this region its characteristic dark colour. In the healthy brain, neuromelanin appears to play a functional role to protect neurons from oxidative load but we have shown that in the Parkinson's disease brain the pigment undergoes structural changes and is associated with aggregation of α-synuclein protein, even early in the disease process. Further, the role of the pigment as a metal binder has also been suggested to underlie the relative vulnerability of these neurons, as changes in metal levels are suggested to be associated with neurodegenerative cascades in Parkinson's disease. While most research to date has focused on the role of iron in these pathways we have recently shown that changes in copper may contribute to neuronal vulnerability in this disorder.

Neuromelanin, neurotransmitter status and brainstem location determine the differential vulnerability of catecholaminergic neurons to mitochondrial DNA deletions

BackgroundDeletions of the mitochondrial DNA (mtDNA) accumulate to high levels in dopaminergic neurons of the substantia nigra pars compacta (SNc) in normal aging and in patients with Parkinson's disease (PD). Human nigral neurons characteristically contain the pigment neuromelanin (NM), which is believed to alter the cellular redox-status. The impact of neuronal pigmentation, neurotransmitter status and brainstem location on the susceptibility to mtDNA damage remains unclear. We quantified mtDNA deletions (ΔmtDNA) in single pigmented and non-pigmented catecholaminergic, as well as non-catecholaminergic neurons of the human SNc, the ventral tegmental area (VTA) and the locus coeruleus (LC), using laser capture microdissection and single-cell real-time PCR.ResultsIn healthy aged individuals, ΔmtDNA levels were highest in pigmented catecholaminergic neurons (25.2 ± 14.9%), followed by non-pigmented catecholamergic (18.0 ± 11.2%) and non-catecholaminergic neurons (12.3 ± 12.3%; p < 0.001). Within the catecholaminergic population, ΔmtDNA levels were highest in dopaminergic neurons of the SNc (33.9 ± 21.6%) followed by dopaminergic neurons of the VTA (21.9 ± 12.3%) and noradrenergic neurons of the LC (11.1 ± 11.4%; p < 0.001). In PD patients, there was a trend to an elevated mutation load in surviving non-pigmented nigral neurons (27.13 ± 16.73) compared to age-matched controls (19.15 ± 11.06; p = 0.052), but levels where similar in pigmented nigral neurons of PD patients (41.62 ± 19.61) and controls (41.80 ± 22.62).ConclusionsCatecholaminergic brainstem neurons are differentially susceptible to mtDNA damage. Pigmented dopaminergic neurons of the SNc show the highest ΔmtDNA levels, possibly explaining the exceptional vulnerability of the nigro-striatal system in PD and aging. Although loss of pigmented noradrenergic LC neurons also is an early feature of PD pathology, mtDNA levels are not elevated in this nucleus in healthy controls. Thus, ΔmtDNA are neither an inevitable consequence of catecholamine metabolism nor a universal explanation for the regional vulnerability seen in PD.

Expression of GDNF receptors GFRα1 and RET is preserved in substantia nigra pars compacta of aging Asian Indians

Glial derived neurotrophic factor (GDNF) protects dopaminergic nigral neurons and may prevent the progression of age-related motor deficits and Parkinson's disease. The multi-component receptor complex which mediates the neuroprotective action of GDNF comprises of GDNF receptor alpha1 (GFRα1), a ligand binding cell surface component and RET receptor tyrosine kinase (RET) the signaling component. The expression of both these receptors in the normally aging human substantia nigra pars compacta (SNpc) needs to be studied since GDNF infusion is being considered for restoration of the lost nigrostriatal function. In the present study, we used unbiased stereology to quantify the number of GFRα1 and RET immunoreactive neurons in human SNpc from 28 weeks of gestation to 88 years ( n = 31). We further determined the levels of immunostaining intensity using densitometric image analysis to measure changes in levels of receptor expression. Here we report that human nigral dopaminergic neurons express GFRα1 and RET receptors at all ages. There was no reduction in the number of neurons expressing these receptors as a function of age. Moreover, there was no age-related decline in immunostaining intensity of both these receptors. It is likely that preservation of GDNF receptors in the nigral neurons is because these receptors are constitutively expressed in the human SNpc and thus it is GDNF responsive thru aging. The sustained receptor protein expression could also be another marker of preserved nigrostriatal function in Asian Indians. The latter possibility explains our earlier observation that the melanized nigral neurons are preserved with age in the Asian Indians.

Gene expression profiling of parkinsonian substantia nigra pars compacta; alterations in ubiquitin-proteasome, heat shock protein, iron and oxidative stress regulated proteins, cell adhesion/cellular matrix and vesicle trafficking genes

Gene expression profiling of human substantia nigra pars compacta (SNpc) from Parkinson's disease (PD) patients, was examined employing high density microarrays. We identified alterations in the expression of 137 genes, with 68 down regulated and 69 up regulated. The down regulated genes belong to signal transduction, protein degradation (e.g. ubiquitin-proteasome subunits), dopaminergic transmission/metabolism, ion transport, protein modification/phosphorylation and energy pathways/glycolysis functional classes. Up-regulated genes, clustered mainly in biological processes involving cell adhesion/cytoskeleton, extracellular matrix components, cell cycle, protein modification/phosphorylation, protein metabolism, transcription and inflammation/stress (e.g. key iron and oxygen sensor EGLN1). One major finding in the present study is the particular decreased expression of SKP1A, a member of the SCF (E3) ligase complex specifically in the substantia nigra (SN) of sporadic parkinsonian patients, which may lead to a wide impairment in the function of an entire repertoire of proteins subjected to regulatory ubiquitination. These findings reveal novel players in the neurodegenerative scenario and provide potential targets for the development of novel drug compounds.

An Altered Histaminergic Innervation of the Substantia Nigra in Parkinson's Disease

The central histaminergic system is one of the subcortical aminergic projection systems involved in several regulatory functions. The central dopaminergic and histaminergic systems interact extensively, but little is known about the histaminergic system in diseases affecting the dopaminergic neurons. The distribution of histaminergic fibers in the substantia nigra (SN) in postmortem brain samples from patients suffering from Parkinson's disease (PD) and normal controls was examined with a specific immunohistochemical method. Direct connections between dopaminergic neurones and histaminergic fibers were observed. Histamine in human SN was stored in fibers and varicosities. Sites of histamine formation were examined by l-histidine decarboxylase in situ hybridization. In both normal and PD brains HDC mRNA was found only in posterior hypothalamus and not in SN. The presence of histaminergic innervation of the human substantia nigra pars compacta (SNc) and reticulata (SNr), paranigral nucleus, radix of oculomotor nerve, and parabrachial pigmented nucleus was demonstrated. The density of histaminergic fibers in the middle portion of SNc and SNr was increased in brains with PD. In PD the morphology of histaminergic fibers was also altered; they were thinner than in controls and had enlarged varicosities. An increase of histaminergic innervation may reflect a compensatory event due to deficiency of, e.g., dopamine or a putative fiber growth inhibitory factor. Whether the changes seen in histaminergic fibers in PD are primary or secondary remains to be investigated.