Postmortem Substantia Nigra Research Articles

Genes for endosomal pH regulators NHE6 and NHE9 are dysregulated in the substantia nigra in Parkinson’s disease

Endosomal acid base balance functions as a master orchestrator within the cell, engaging with many cellular pathways to maintain homeostasis. Mutations in the endosomal pH regulator Na+/H+ exchanger NHE6 may disrupt this delicate balancing act and cause monogenic Parkinsonism. Here, gene expression studies in post-mortem substantia nigra of Parkinson’s disease (PD) patients and normal controls were performed to investigate whether NHE6 represents a pathophysiological link between monogenic and sporadic PD. The substantia nigra in PD displayed down-regulation of NHE6, coincident with a loss of expression of several SNARE signalling pathway members, suggesting impaired membrane fusion and vesicle-recycling. Increased abundance of related NHE9 was also identified in the parkinsonian nigra that could reflect compensatory changes or be a consequence of neuronal dysfunction. The current model suggests the possibility that neurons expressing low levels of NHE6 are more susceptible to injury in PD, potentially directly contributing to the loss of nigral dopaminergic neurons and the genesis of the disease. These results have important implications for disease-modifying therapies as they suggest that endosomal pH correctors, including epigenetic modifiers that regulate NHE6 expression, may be beneficial for PD. Thus, aberrant endosomal acidification in the nigrostriatal pathway is a possible unifying pathomechanism in both monogenic and sporadic PD, with implications for understanding and treating this disorder. Replication of these observations in the post-mortem brains of Alzheimer’s disease and frontotemporal dementia patients supports a model of conserved mechanisms underlying injury and death of neurons.

The contribution of inflammatory astrocytes to BBB impairments in a brain-chip model of Parkinson’s disease

Astrocyte dysfunction has previously been linked to multiple neurodegenerative disorders including Parkinson’s disease (PD). Among their many roles, astrocytes are mediators of the brain immune response, and astrocyte reactivity is a pathological feature of PD. They are also involved in the formation and maintenance of the blood-brain barrier (BBB), but barrier integrity is compromised in people with PD. This study focuses on an unexplored area of PD pathogenesis by characterizing the interplay between astrocytes, inflammation and BBB integrity, and by combining patient-derived induced pluripotent stem cells with microfluidic technologies to generate a 3D human BBB chip. Here we report that astrocytes derived from female donors harboring the PD-related LRRK2 G2019S mutation are pro-inflammatory and fail to support the formation of a functional capillary in vitro. We show that inhibition of MEK1/2 signaling attenuates the inflammatory profile of mutant astrocytes and rescues BBB formation, providing insights into mechanisms regulating barrier integrity in PD. Lastly, we confirm that vascular changes are also observed in the human postmortem substantia nigra of both males and females with PD.

NF-κB/c-Rel DNA-binding is reduced in substantia nigra and peripheral blood mononuclear cells of Parkinson's disease patients

Although Parkinson's disease (PD) key neuropathological hallmarks are well known, the underlying pathogenic mechanisms of the disease still need to be elucidated to identify innovative disease-modifying drugs and specific biomarkers. NF-κB transcription factors are involved in regulating several processes associated with neurodegeneration, such as neuroinflammation and cell death, that could be related to PD pathology. NF-κB/c-Rel deficient (c-rel−/−) mice develop a progressive PD-like phenotype. The c-rel−/− mice present both prodromal and motor symptoms as well as key neuropathological features, including nigrostriatal dopaminergic neurons degeneration, accumulation of pro-apoptotic NF-κB/RelA acetylated at the lysine 310 residue (Ac-RelA(lys310)) and progressive caudo-rostral brain deposition of alpha-synuclein. c-Rel inhibition can exacerbate MPTP-induced neurotoxicity in mice. These findings support the claim that misregulation of c-Rel protein may be implicated in PD pathophysiology. In this study, we aimed at evaluating c-Rel levels and DNA-binding activity in human brains and peripheral blood mononuclear cells (PBMCs) of sporadic PD patients. We analyzed c-Rel protein content and activity in frozen substantia nigra (SN) samples from post-mortem brains of 10 PD patients and 9 age-matched controls as well as in PBMCs from 72 PD patients and 40 age-matched controls. c-Rel DNA-binding was significantly lower and inversely correlated with Ac-RelA(lys310) content in post-mortem SN of sporadic PD cases, when compared to healthy controls. c-Rel DNA-binding activity was also reduced in PBMCs of followed-up PD subjects. The decrease of c-Rel activity in PBMCs from PD patients appeared to be independent from dopaminergic medication or disease progression, as it was evident even in early stage, drug-naïve patients. Remarkably, the levels of c-Rel protein were comparable in PD and control subjects, pointing out a putative role for post-translational modifications of the protein in c-Rel dysfunctions.These findings support that PD is characterized by the loss of NF-κB/c-Rel activity that potentially has a role in PD pathophysiology. Future studies will be aimed at addressing whether the reduction of c-Rel DNA-binding could constitute a novel biomarker for PD.

DOPA pheomelanin is increased in nigral neuromelanin of Parkinson’s disease

Neuromelanin (NM) in dopaminergic neurons of human substantia nigra (SN) has a melanic component that consists of pheomelanin and eumelanin moieties and has been proposed as a key factor contributing to dopaminergic neuron vulnerability in Parkinson’s disease (PD). While eumelanin is considered as an antioxidant, pheomelanin and related oxidative stress are associated with compromised drug and metal ion binding and melanoma risk. Using postmortem SN from patients with PD or Alzheimer’s disease (AD) and unaffected controls, we identified increased L-3,4-dihydroxyphenylalanine (DOPA) pheomelanin and increased ratios of dopamine (DA) pheomelanin markers to DA in PD SN compared to controls. Eumelanins derived from both DOPA and DA were reduced in PD group. In addition, we report an increase in DOPA pheomelanin relative to DA pheomelanin in PD SN. In AD SN, we observed unaltered melanin markers despite reduced DOPA compared to controls. Furthermore, synthetic DOPA pheomelanin induced neuronal cell death in vitro while synthetic DOPA eumelanin showed no significant effect on cell viability. Our findings provide insights into the different roles of pheomelanin and eumelanin in PD pathophysiology. We anticipate our study will lead to further investigations on pheomelanin and eumelanin individually as biomarkers and possibly therapeutic targets for PD.

Evidence for upregulation of excitatory synaptic transmission in the substantia nigra in Schizophrenia: a postmortem ultrastructural study.

The dopamine hypothesis of schizophrenia suggests that psychotic symptoms originate from dysregulation of dopaminergic activity, which may be controlled by upstream innervation. We hypothesized that we would find anatomical evidence for the hyperexcitability seen in the SN. We examined and quantified synaptic morphology, which correlates with function, in the postmortem substantia nigra (SN) from 15 schizophrenia and 12 normal subjects. Synapses were counted using stereological techniques and classified based on the morphology of the post-synaptic density (PSD) and the presence or absence of a presynaptic density. The density and proportion of excitatory synapses was higher in the schizophrenia group than in controls, while the proportion (but not density) of inhibitory synapses was lower. We also detected in the schizophrenia group an increase in density of synapses with a PSD of intermediate thickness, which may represent excitatory synapses. The density of synapses with presynaptic densities was similar in both groups. The density of synapses with mixed morphologies was higher in the schizophrenia group than in controls. The human SN contains atypical synaptic morphology. We found an excess amount and proportion of excitatory synapses in the SN in schizophrenia that could result in hyperactivity and drive the psychotic symptoms of schizophrenia. The sources of afferent excitatory inputs to the SN arise from the subthalamic nucleus, the pedunculopontine nucleus, and the ventral tegmental area (VTA), areas that could be the source of excess excitation. Synapses with mixed morphologies may represent inputs from the VTA, which release multiple transmitters.

Identification of Immune-Related Hub Genes in Parkinson’s Disease

Background: Parkinson’s disease (PD) is a common, age-related, and progressive neurodegenerative disease. Growing evidence indicates that immune dysfunction plays an essential role in the pathogenic process of PD. The objective of this study was to explore potential immune-related hub genes and immune infiltration patterns of PD. Method: The microarray expression data of human postmortem substantia nigra samples were downloaded from GSE7621, GSE20141, and GSE49036. Key module genes were screened via weighted gene coexpression network analysis, and immune-related genes were intersected to obtain immune-key genes. Functional enrichment analysis was performed on immune-key genes of PD. In addition to, immune infiltration analysis was applied by a single-sample gene set enrichment analysis algorithm to detect differential immune cell types in the substantia nigra between PD samples and control samples. Least absolute shrinkage and selection operator analysis was performed to further identify immune-related hub genes for PD. Receiver operating characteristic curve analysis of the immune-related hub genes was used to differentiate PD patients from healthy controls. Correlations between immune-related hub genes and differential immune cell types were assessed. Result: Our findings identified four hub genes (SLC18A2, L1CAM, S100A12, and CXCR4) and seven immune cell types (neutrophils, T follicular helper cells, myeloid-derived suppressor cells, type 1 helper cells, immature B cells, immature dendritic cells, and CD56 bright natural killer cells). The area under the curve (AUC) value of the four-gene-combined model was 0.92. The AUC values of each immune-related hub gene (SLC18A2, L1CAM, S100A12, and CXCR4) were 0.81, 0.78, 0.78, and 0.76, respectively. Conclusion: In conclusion, SLC18A2, L1CAM, S100A12, and CXCR4 were identified as being associated with the pathogenesis of PD and should be further researched.

Ecto-GPR37: a potential biomarker for Parkinson\u2019s disease

Objectiveα-Synuclein has been studied as a potential biomarker for Parkinson’s disease (PD) with no concluding results. Accordingly, there is an urgent need to find out reliable specific biomarkers for PD. GPR37 is an orphan G protein-coupled receptor that toxically accumulates in autosomal recessive juvenile parkinsonism. Here, we investigated whether GPR37 is upregulated in sporadic PD, and thus a suitable potential biomarker for PD.MethodsGPR37 protein density and mRNA expression in postmortem substantia nigra (SN) from PD patients were analysed by immunoblot and RT-qPCR, respectively. The presence of peptides from the N-terminus-cleaved domain of GPR37 (i.e. ecto-GPR37) in human cerebrospinal fluid (CSF) was determined by liquid chromatography-mass spectrometric analysis. An engineered in-house nanoluciferase-based immunoassay was used to quantify ecto-GPR37 in CSF samples from neurological control (NC) subjects, PD patients and Alzheimer’s disease (AD) patients.ResultsGPR37 protein density and mRNA expression were significantly augmented in sporadic PD. Increased amounts of ecto-GPR37 peptides in the CSF samples from PD patients were identified by mass spectrometry and quantified by the in-house ELISA method. However, the CSF total α-synuclein level in PD patients did not differ from that in NC subjects. Similarly, the cortical GPR37 mRNA expression and CSF ecto-GPR37 levels in AD patients were also unaltered.ConclusionGPR37 expression is increased in SN of sporadic PD patients. The ecto-GPR37 peptides are significantly increased in the CSF of PD patients, but not in AD patients. These results open perspectives and encourage further clinical studies to confirm the validity and utility of ecto-GPR37 as a potential PD biomarker.

MRI T2 and T2* relaxometry to visualize neuromelanin in the dorsal substantia nigra pars compacta

Visualizing gradual changes in neuromelanin distribution within the substantia nigra is an important metric used to monitor the progression of Parkinsonism. This study aimed to identify the origin of the mismatch region between magnetic resonance transverse relaxation times (T2 and T2*) in the substantia nigra and investigate its feasibility and implications for in vivo detection of neuromelanin as a clinical biomarker. The relationships between neuromelanin distribution assessed by histological staining and the area of T2 and T2* mismatch determined by high- and low-resolution magnetic resonance relaxometry at 7T were directly compared in two normal and one depigmented substantia nigra collected at postmortem. In vivo feasibility of assessing T2 and T2* mismatch, clinically, was investigated using 3T magnetic resonance imaging. In the normal postmortem substantia nigra tissue, the T2 and T2* mismatch region exhibiting a linear pattern was strongly colocalized with neuromelanin distribution along the dorsal substantia nigra pars compacta, but a negligible amount of dorsal mismatch was observed in the depigmented brain. The regions of T2 and T2* mismatch from MRI, neuromelanin pigments from histology, and elevated iron signals from mass spectrometry were spatially overlapped for a normal postmortem brain. In preliminary in vivo studies, a similar, linear T2 and T2* mismatch region was observed in the dorsal area of the substantia nigra in eight normal subjects; this mismatch was significantly obscured in eight Parkinson’s disease patients. The length of the dorsal linear mismatch line based on the T2*-T2 mask was significantly shorter in the Parkinson’s disease patients compared to normal controls; this result was corroborated by reduced striatal uptake of [18F] FP-CIT dopamine transporters assessed by positron emission tomography scans. In conclusion, the measurement of T2 and T2* mismatch could serve as a complementary imaging biomarker to visualize the dorsal region of the substantia nigra pars compacta, which contains large amounts of neuromelanin.

Reduced sphingolipid hydrolase activities, substrate accumulation and ganglioside decline in Parkinson\u2019s disease

BackgroundHaploinsufficiency in the Gaucher disease GBA gene, which encodes the lysosomal glucocerebrosidase GBA, and ageing represent major risk factors for developing Parkinson’s disease (PD). Recently, more than fifty other lysosomal storage disorder gene variants have been identified in PD, implicating lysosomal dysfunction more broadly as a key risk factor for PD. Despite the evidence of multiple lysosomal genetic risks, it remains unclear how sphingolipid hydrolase activities, other than GBA, are altered with ageing or in PD. Moreover, it is not fully known if levels of glycosphingolipid substrates for these enzymes change in vulnerable brain regions of PD. Finally, little is known about the levels of complex gangliosides in substantia nigra which may play a significant role in ageing and PD.MethodsTo study sphingolipid hydrolase activities and glycosphingolipid expression in ageing and in PD, two independent cohorts of human substantia nigra tissues were obtained. Fluorescent 4-methylumbelliferone assays were used to determine multiple enzyme activities. The lysosomal GBA and non-lysosomal GBA2 activities were distinguished using the inhibitor NB-DGJ. Sensitive and quantitative normal-phase HPLC was performed to study glycosphingolipid levels. In addition, glycosphingolipid levels in cerebrospinal fluid and serum were analysed as possible biomarkers for PD.ResultsThe present study demonstrates, in two independent cohorts of human post-mortem substantia nigra, that sporadic PD is associated with deficiencies in multiple lysosomal hydrolases (e.g. α-galactosidase and β-hexosaminidase), in addition to reduced GBA and GBA2 activities and concomitant glycosphingolipid substrate accumulation. Furthermore, the data show significant reductions in levels of complex gangliosides (e.g. GM1a) in substantia nigra, CSF and serum in ageing, PD, and REM sleep behaviour disorder, which is a strong predictor of PD.ConclusionsThese findings conclusively demonstrate reductions in GBA activity in the parkinsonian midbrain, and for the first time, reductions in the activity of several other sphingolipid hydrolases. Furthermore, significant reductions were seen in complex gangliosides in PD and ageing. The diminished activities of these lysosomal hydrolases, the glycosphingolipid substrate accumulation, and the reduced levels of complex gangliosides are likely major contributors to the primary development of the pathology seen in PD and related disorders with age.

Evidence for altered excitatory and inhibitory tone in the post-mortem substantia nigra in schizophrenia

Objectives: The substantia nigra (SN) receives glutamatergic and GABAergic inputs that regulate dopaminergic neuronal activity. Imaging studies have shown hyperactivity of the SN in schizophrenia (SZ) patients. We examined neurochemically defined inputs to the SN, synaptic density, and neuromelanin content that might contribute to or reflect this hyperexcitability.Methods: Glutamatergic axon terminals were identified by the immunohistochemical localisation of vGLUT1 and vGLUT2; GABA inputs were identified by the immunohistochemical localisation of GAD67. Neuromelanin granules are visible in unstained sections and thus were assessed in unstained sections. Optical densitometry was measured to assess the density of vGLUT1, vGLUT2 or GAD67 immunolabelled axon terminals and neuromelanin granules. Electron microscopy was used to quantify synaptic and mitochondrial density.Results: Compared to controls, SZ subjects had nonsignificant trends toward a decrease in vGLUT1, and an increase in both vGLUT2 and GAD67. vGLUT1 was negatively correlated with GAD67 in normal controls (NCs) and positively correlated in SZ subjects. A correlation of coefficient analysis showed a significant difference between the negative correlation in NCs and the positive correlation in SZ subjects. Frequency histograms showed the distribution of neuromelanin density was different in SZ subjects compared to NCs. Synaptic density data showed a decrease in inhibitory synapses in SZ subjects. Mitochondrial density was normal in SZ subjects.Conclusions: Synaptic density alterations and the lack of a positive correlation between GAD67 and vGLUT1 could contribute to hyperactivity in the SN.

S-Nitrosylation of Divalent Metal Transporter 1 Enhances Iron Uptake to Mediate Loss of Dopaminergic Neurons and Motoric Deficit.

Elevated iron deposition has been reported in Parkinson's disease (PD). However, the route of iron uptake leading to high deposition in the substantia nigra is unresolved. Here, we show a mechanism in enhanced Fe2+ uptake via S-nitrosylation of divalent metal transporter 1 (DMT1). While DMT1 could be S-nitrosylated by exogenous nitric oxide donors, in human PD brains, endogenously S-nitrosylated DMT1 was detected in postmortem substantia nigra. Patch-clamp electrophysiological recordings and iron uptake assays confirmed increased Mn2+ or Fe2+ uptake through S-nitrosylated DMT1. We identified two major S-nitrosylation sites, C23 and C540, by mass spectrometry, and DMT1 C23A or C540A substitutions abolished nitric oxide (NO)-mediated DMT1 current increase. To evaluate in vivo significance, lipopolysaccharide (LPS) was stereotaxically injected into the substantia nigra of female and male mice to induce inflammation and production of NO. The intranigral LPS injection resulted in corresponding increase in Fe2+ deposition, JNK activation, dopaminergic neuronal loss and deficit in motoric activity, and these were rescued by the NO synthase inhibitor l-NAME or by the DMT1-selective blocker ebselen. Lentiviral knockdown of DMT1 abolished LPS-induced dopaminergic neuron loss.SIGNIFICANCE STATEMENT Neuroinflammation and high cytoplasmic Fe2+ levels have been implicated in the initiation and progression of neurodegenerative diseases. Here, we report the unexpected enhancement of the functional activity of transmembrane divalent metal transporter 1 (DMT1) by S-nitrosylation. We demonstrated that S-nitrosylation increased DMT1-mediated Fe2+ uptake, and two cysteines were identified by mass spectrometry to be the sites for S-nitrosylation and for enhanced iron uptake. One conceptual advance is that while DMT1 activity could be increased by external acidification because the gating of the DMT1 transporter is proton motive, we discovered that DMT1 activity could also be enhanced by S-nitrosylation. Significantly, lipopolysaccharide-induced nitric oxide (NO)-mediated neuronal death in the substantia nigra could be ameliorated by using l-NAME, a NO synthase inhibitor, or by ebselen, a DMT1-selective blocker.

Upregulation of neuronal astrocyte elevated gene-1 protects nigral dopaminergic neurons in vivo

The role of astrocyte elevated gene-1 (AEG-1) in nigral dopaminergic (DA) neurons has not been studied. Here we report that the expression of AEG-1 was significantly lower in DA neurons in the postmortem substantia nigra of patients with Parkinson’s disease (PD) compared to age-matched controls. Similarly, decreased AEG-1 levels were found in the 6-hydroxydopamine (6-OHDA) mouse model of PD. An adeno-associated virus-induced increase in the expression of AEG-1 attenuated the 6-OHDA-triggered apoptotic death of nigral DA neurons. Moreover, the neuroprotection conferred by the AEG-1 upregulation significantly intensified the neurorestorative effects of the constitutively active ras homolog enriched in the brain [Rheb(S16H)]. Collectively, these results demonstrated that the sustained level of AEG-1 as an important anti-apoptotic factor in nigral DA neurons might potentiate the therapeutic effects of treatments, such as Rheb(S16H) administration, on the degeneration of the DA pathway that characterizes PD.

Specific visualization of neuromelanin-iron complex and ferric iron in the human post-mortem substantia nigra using MR relaxometry at 7T

Neuromelanin (NM) is an endogenous iron chelating molecule of pigmented neurons in the human substantia nigra (SN). Along with the increase in iron deposition, the reduction in NM-containing dopaminergic neurons and the variation of iron load on NM are generally considered to be important factors participating to pathogenesis of Parkinson's disease (PD). The aim of this study was to non-invasively delineate the spatial distributions of paramagnetic magnetic susceptibility perturbers, such as NM-iron complex and ferric iron in SN. Multiple quantitative MR parameters of T1, T2, T2*, susceptibility weighted image (SWI), quantitative susceptibility map (QSM), and T1 weighted image with magnetization transfer (MT) effects were acquired for six post-mortem SN samples without a history of neurological disease. Co-registered quantitative histological validations were performed to identify and correlate NM pigments, iron deposits, and myelin distributions with respect to associated MR parameters. The regions with NM pigments and iron deposits showed positive magnetic susceptibility (paramagnetic) values, while myelinated areas showed negative magnetic susceptibility (diamagnetic) values from the QSM. The region of reduced T2 values in SN mostly coincided with high iron deposits, but not necessarily with the NM pigments. The correlations between T2*/T2 (or T2*/T22) values and NM pigments were higher than those between T2* values and NM pigments, due to the effective size differences between NM-iron complex and ferric iron. Consequently, separate segmentations of ferric iron from the T2 map and NM-iron complex from the T2*/T2 map (or T2*/T22 map) were possible with the boundary of the SN determined from the T1 weighted image.

SA100. Abnormalities in Copper Transporters ATP7A and CTR1 in Postmortem Substantia Nigra in Schizophrenia

SA100. Abnormalities in Copper Transporters ATP7A and CTR1 in Postmortem Substantia Nigra in Schizophrenia

SU98. Glutamate and GABA in the Substantia Nigra in Postmortem Brain

Abstract Background: Schizophrenia (SZ) is a mental illness that has positive, negative, and cognitive symptoms. The substantia nigra (SN) is one of the largest dopaminergic nuclei in the brain and projects to the striatum which is the primary locus for action of antipsychotic drugs. The SN receives both glutamatergic and GABAergic inputs that regulate the dopaminergic neuronal activity. In recent years, imaging studies that have shown hyperactivity of the SN in patients with SZ, suggesting that dysregulation of the SN may play of role in the increased amount of dopamine in the striatum in these patients. Methods: Our study examined neurochemically defined inputs to the SN. The postmortem SN that was used was collected from the Maryland Brain Collection. There were 11 cases total that were examined in this study with schizophrenia cases (n = 6) being compared to matched normal control cases (SZ) (n = 5). In this study, immunohistochemistry was performed with 3 different antibodies: vGLUT1, vGLUT2, and GAD67, which labels glutamatergic (vGLUT1 and vGLUT2) and GABAergic (GAD67) projections to the SN. VGLUT1 labels projections coming from cortical and hippocampal regions while vGLUT2 labels projections coming from subcortical regions such as the subthalamic nucleus and the pedunculopontine nucleus. The stained tissues were mounted, cover slipped, and the entirety of the SN was imaged using a light box. Three to 4 sections were analyzed per case. Using a random coordinate generator, 20 areas were selected from each section from which 25 photos were taken for each; optical densitometry (OD) analysis was performed using ImageJ. Results: The mean OD values for vGLUT1 were 16.1 ± 5.6 (NC) and 11.6 ± 6.6 (SZ); a 28% decrease for SZ. The mean OD values for vGLUT2 were 35.8 ± 3.9 (NC) and 41.1 ± 6.7 (SZ), a 15% increase for SZ. The mean OD values for GAD67 were 26.5 ± 13.4 (NC) and 32.2 ± 20.0 (SZ), a 21% increase for SZ. Conclusion: None of these changes reached statistical significance, due in part to the small sample size and great variation in results in the SZ group. A previous study from our lab showed changes in protein levels in the SN that were dependent on medication status. Ongoing studies include an electron microscopic analysis of mitochondria per terminal in the SZ, to determine if there are compromised energy demands in specific subsets of terminals.

The Neuromelanin-related T2* Contrast in Postmortem Human Substantia Nigra with 7T MRI.

High field magnetic resonance imaging (MRI)-based delineation of the substantia nigra (SN) and visualization of its inner cellular organization are promising methods for the evaluation of morphological changes associated with neurodegenerative diseases; however, corresponding MR contrasts must be matched and validated with quantitative histological information. Slices from two postmortem SN samples were imaged with a 7 Tesla (7T) MRI with T1 and T2* imaging protocols and then stained with Perl’s Prussian blue, Kluver-Barrera, tyrosine hydroxylase, and calbindin immunohistochemistry in a serial manner. The association between T2* values and quantitative histology was investigated with a co-registration method that accounts for histology slice preparation. The ventral T2* hypointense layers between the SNr and the crus cerebri extended anteriorly to the posterior part of the crus cerebri, which demonstrates the difficulty with an MRI-based delineation of the SN. We found that the paramagnetic hypointense areas within the dorsolateral SN corresponded to clusters of neuromelanin (NM). These NM-rich zones were distinct from the hypointense ventromedial regions with high iron pigments. Nigral T2* imaging at 7T can reflect the density of NM-containing neurons as the metal-bound NM macromolecules may decrease T2* values and cause hypointense signalling in T2* imaging at 7T.

In-vivo evidence that high mobility group box 1 exerts deleterious effects in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine model and Parkinson's disease which can be attenuated by glycyrrhizin

High-mobility group box 1 (HMGB1) is a nuclear and cytosolic protein that is released during tissue damage from immune and non-immune cells — including microglia and neurons. HMGB1 can contribute to progression of numerous chronic inflammatory and autoimmune diseases which is mediated in part by interaction with the receptor for advanced glycation endproducts (RAGE). There is increasing evidence from in vitro studies that HMGB1 may link the two main pathophysiological components of Parkinson's disease (PD), i.e. progressive dopaminergic degeneration and chronic neuroinflammation which underlie the mechanistic basis of PD progression.Analysis of tissue and biofluid samples from PD patients, showed increased HMGB1 levels in human postmortem substantia nigra specimens as well as in the cerebrospinal fluid and serum of PD patients. In a mouse model of PD induced by sub-acute administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), systemic administration of neutralizing antibodies to HMGB1 partly inhibited the dopaminergic cell death, and reduced the increase of RAGE and tumour necrosis factor-alpha. The small natural molecule glycyrrhizin, a component from liquorice root which can directly bind to HMGB1, both suppressed MPTP-induced HMGB1 and RAGE upregulation while reducing MPTP-induced dopaminergic cell death in a dose dependent manner.These results provide first in vivo evidence that HMGB1 serves as a powerful bridge between progressive dopaminergic neurodegeneration and chronic neuroinflammation in a model of PD, suggesting that HMGB1 is a suitable target for neuroprotective trials in PD.

TRPV1 on astrocytes rescues nigral dopamine neurons in Parkinson's disease via CNTF.

Currently there is no neuroprotective or neurorestorative therapy for Parkinson's disease. Here we report that transient receptor potential vanilloid 1 (TRPV1) on astrocytes mediates endogenous production of ciliary neurotrophic factor (CNTF), which prevents the active degeneration of dopamine neurons and leads to behavioural recovery through CNTF receptor alpha (CNTFRα) on nigral dopamine neurons in both the MPP(+)-lesioned or adeno-associated virus α-synuclein rat models of Parkinson's disease. Western blot and immunohistochemical analysis of human post-mortem substantia nigra from Parkinson's disease suggests that this endogenous neuroprotective system (TRPV1 and CNTF on astrocytes, and CNTFRα on dopamine neurons) might have relevance to human Parkinson's disease. Our results suggest that activation of astrocytic TRPV1 activates endogenous neuroprotective machinery in vivo and that it is a novel therapeutic target for the treatment of Parkinson's disease.

Identification of CB2 receptors in human nigral neurons that degenerate in Parkinson’s disease

It is well-demonstrated that cannabinoid CB2 receptors located in glial cells are up-regulated in neurodegenerative disorders serving as a target to control glial influences to neurons. Recent evidence indicates that CB2 receptors may be also located in certain neuronal subpopulations and serve as a marker of neuronal losses. We investigated this possibility in the post-mortem substantia nigra of Parkinson’s disease (PD) patients and controls. Immunostaining for the CB2 receptor was found in tyrosine hydroxylase-positive neurons in the substantia nigra, a fact confirmed with double-staining analyses. The signal was found in controls but also in PD patients, in which CB2 receptor labelling was significantly lower, in parallel to the losses of these neurons experienced in the disease. These data show for the first time that CB2 receptors are located in tyrosine hydroxylase-containing neurons in the substantia nigra at levels significantly lower in PD patients compared to controls.

Correlation of striatal dopamine transporter imaging with post mortem substantia nigra cell counts.

Dopamine transporter imaging is widely used for the differential diagnosis of parkinsonism. Only limited data are available on the relationship between striatal dopamine transporter binding and dopaminergic cell loss in the substantia nigra (SN). We analyzed postmortem SN cell counts in patients who had previously undergone dopamine transporter single-photon emission computed tomography (SPECT). Pathological diagnoses included Parkinson's disease (n = 1), dementia with Lewy bodies (n = 2), multiple system atrophy (n = 1), corticobasal degeneration (n = 2), atypical parkinsonism with multiple pathological conditions (n = 1), Alzheimer's disease (n = 1), and Creutzfeldt-Jakob disease (n = 1). [(12) (3) I]β-CIT SPECT had been performed in all subjects using a standardized protocol on the same triple-head gamma camera. The density of neuromelanin-containing and tyrosine hydroxylase-positive substantia nigra neurons/mm(2) was evaluated in paraffin-embedded tissue sections by morphometric methods. Mean disease duration at the time of dopamine transporter imaging was 2.3 years, and the mean interval from imaging to death was 29.3 months (range, 4-68 months). Visual analysis of dopamine transporter images showed reduced striatal uptake in all seven patients with neurodegenerative parkinsonism, but not in Alzheimer's and Creutzfeldt-Jakob disease cases. Averaged [(right+left)/2] striatal uptake was highly correlated with averaged SN cell counts (rs = 0.98, P < 0.0005 for neuromelanin- and rs = 0.96, P < 0.0005 for tyrosine hydroxylase-positive cells). Similar strong correlations were found in separate analyses for the right and left sides. Striatal dopamine transporter binding highly correlated with postmortem SN cell counts, confirming the validity of dopamine transporter imaging as an excellent in vivo marker of nigrostriatal dopaminergic degeneration.