Mössbauer spectroscopy and ELISA studies reveal differences between Parkinson's disease and control substantia nigra

The possible role of iron in the degeneration of nervous cells in Parkinson's disease (PD) was studied with the use of M?ssbauer spectroscopy (MS) and enzyme-linked immunoabsorbent assay (ELISA). M?ssbauer data were obtained at 90 and 4.1 K from 21 samples of control and 9 samples of parkinsonian substantia nigra (SN). M?ssbauer spectra were very similar to those observed in ferritin. Small differences were detected between the spectra obtained from PD and from control SN, and could be due to a slight difference in the composition of the ferritin-like iron cores or due to the presence of about 8% of non-ferritin-like iron in parkinsonian SN. ELISA studies from 11 controls and 6 parkinsonian SN showed a decrease in the concentration of L-chains in wet tissues of PD-SN compared to control SN. The decrease in the amount of L subunits may correspond to a decreased ability of this ferritin to keep iron in a safe form. Iron released from ferritin or neuromelanin (NM) may be the source of such iron, which may cause the difference in the M?ssbauer spectra and may trigger oxidative stress leading to cell death.     

Is the Vulnerability of Neurons in the Substantia Nigra of Patients with Parkinson's Disease Related to Their Neuromelanin Content?

The contribution of neuromelanin (NM) to the pathogenesis of Parkinson's disease (PD) has long been suspected. In particular, a correlation has been reported between the estimated cell loss in the mesencephalic dopaminergic cell groups and the percentage of NM-pigmented neurons in these cell groups. To test whether the amount of pigment per cell is a critical factor or whether the presence of NM within a neuron is sufficient to account for the degeneration of dopaminergic neurons, the NM content was measured in each neuron from representative sections throughout the ventral mesencephalon of four controls subjects and four patients with PD. Intraneuronal NM was quantified by a densitometric method, using known amounts of synthetic melanin as standards. In control brains, the distribution of melanized neurons in the nigral complex showed a high proportion of lightly melanized neurons in the ventral tegmental area and the pars alpha and gamma of the substantia nigra (SN), whereas heavily melanized neurons were mostly located in the pars beta and lateralis of the SN. An inverse relationship was observed between the percentage of surviving neurons in PD compared with controls and the amount of NM they contain, suggesting that the vulnerability of the dopaminergic neurons is related to their NM content. Factors other than NM may be involved in the differential vulnerability of catecholaminergic neurons in PD. In particular, the constant topography of the cell loss suggests that cell position within the nigral complex is a key factor.     

Mass Spectrometry–Based Proteomics Analysis of Human Substantia Nigra From Parkinson's Disease Patients Identifies Multiple Pathways Potentially Involved in the Disease

Parkinson's disease (PD) is the second most prevalent neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra (SN) of the brain. Despite decades of studies, the precise pathogenic mechanism of PD is still elusive. An unbiased proteomic analysis of PD patient’s brain allows the identification of critical proteins and molecular pathways that lead to dopamine cell death and α-synuclein deposition and the resulting devastating clinical symptoms. In this study, we conducted an in-depth proteome analysis of human SN tissues from 15 PD patients and 15 healthy control individuals combining Orbitrap mass spectrometry with the isobaric tandem mass tag–based multiplexing technology. We identified 10,040 proteins with 1140 differentially expressed proteins in the SN of PD patients. Pathway analysis showed that the ribosome pathway was the most enriched one, followed by gamma-aminobutyric acidergic synapse, retrograde endocannabinoid signaling, cell adhesion molecules, morphine addiction, Prion disease, and PD pathways. Strikingly, the majority of the proteins enriched in the ribosome pathway were mitochondrial ribosomal proteins (mitoribosomes). The subsequent protein–protein interaction analysis and the weighted gene coexpression network analysis confirmed that the mitoribosome is the most enriched protein cluster. Furthermore, the mitoribosome was also identified in our analysis of a replication set of ten PD and nine healthy control SN tissues. This study provides potential disease pathways involved in PD and paves the way to study further the pathogenic mechanism of PD.     

Identification of L-ferritin in Neuromelanin Granules of the Human Substantia Nigra: A TARGETED PROTEOMICS APPROACH*

In the pigmented dopaminergic neurons of the human substantia nigra pars compacta the system relevant in iron storage is the polymer neuromelanin (NM). Although in most cells this function is mainly accomplished by ferritin, this protein complex appears not to be expressed in NM-containing neurons. Nevertheless the conceivable presence of iron-storing proteins as part of the NM granules has recently been discussed on the basis of Mössbauer spectroscopy and synchrotron x-ray microspectroscopy. Intriguingly by combining subcellular fractionation of NM granules, peptide sequencing via tandem mass spectrometry, and the additional confirmation by multiple reaction monitoring and immunogold labeling for electron microscopy, L-ferritin could now be unambiguously identified and localized in NM granules for the first time. This finding not only supports direct evidence for a regulatory role of L-ferritin in neuroectodermal cell pigmentation but also integrates a new player within a complicated network governing iron homeostasis in the dopamine neurons of the human substantia nigra. Thus our finding entails far reaching implications especially when considering etiopathogenetic aspects of Parkinson disease.Neuromelanin (NM)¹ is a dark colored polymeric pigment produced in specific populations of catecholaminergic neurons in the brain (1). Unlike peripheral melanins, which are produced in specialized cells called melanocytes and may be transferred to other cell types, NM granules are believed to be stored in the neurons in which they are produced. NM granules display a unique, more heterogeneous appearance compared with peripheral melanins. Further unlike melanin, NM is traditionally thought to result from a non-enzymatic synthesis pathway with no known pathway for NM catabolism. More recent data, however, are indicative of some regulation of NM synthesis and turnover (1).NM appears in greatest quantities in the human brain and in lesser amounts in some other non-human primates but is absent from the brain of many lower species. Interest in this pigment has seen a resurgence in recent years because of a hypothesized link between NM and the especial vulnerability of NM-containing neurons of the substantia nigra pars compacta (SN) for cell death in Parkinson disease (PD) (2, 3). In particular, the interaction between iron and NM has been a focus of research (4–8) because a marked accumulation of iron related to disease severity is reported in the parkinsonian SN (9–11). The cellular location of this apparent increase in iron is unclear, but a variety of changes in iron regulatory systems occur in PD (12–15).A potential candidate for intraneuronal iron homeostasis in the SN, however, is NM. NM is able to bind a variety of metals; 7% (w/w) of isolated NM is reported to consist of iron, copper, zinc, manganese, and chromium (16, 17). Iron binding studies using NM isolated from the human SN demonstrated that NM contains high (K_D = 7.18 ± 1.08 nm) and low affinity binding sites (K_D = 94.31 ± 6.55 nm) for Fe(III) (18). Our recent data showed that a pure Fe(III) signal can be measured from intact frozen SN tissue using Mössbauer spectroscopy (18). These data indicated that iron is directly bound to NM granules in the SN (4, 16, 19) and that this signal is increased in PD (20). In addition, Mössbauer spectroscopy showed that iron binding sites in NM isolated from the human SN are similar to those of human ferritin and hemosiderin (21). Similar results were also reported recently in whole neurons from formalin-fixed and paraffin-embedded human SN sections using synchrotron chemical x-ray microscopy (22). Because ferritin, the main iron storage protein, is primarily located in glia rather than in neurons (23), it seems unlikely that it could regulate neuronal iron levels, and until today the exact iron storing mechanism in the NM-containing neurons of the SN was unknown.The aim of the present study was thus to find direct evidence for the presence of L-ferritin in NM granules isolated from human post-mortem tissue of subjects with no history of neurological, neurodegenerative, or psychiatric diseases by using a targeted MS-based approach. Recently our group reported a method for the isolation of intact NM granules from the human SN to carry out the first protein profile of these organelles (24). The major findings were the identifications of numerous proteins closely associated with lysosome-related organelles originating from the endosomal system (24, 25). In our present study, we report for the first time the identification of L-ferritin as a component of NM granules, pointing to a ferritin-based iron storage mechanism in the NM-containing neurons of the SN, by using an approach combining one-dimensional (1-D) SDS-PAGE, reversed-phase nano-HPLC electrospray ionization tandem mass spectrometry (nano-LC-ESI-MS/MS and nano-LC-ESI-multiple reaction monitoring (MRM)-MS/MS), Western blot analysis, and immunotransmission electron microscopy.     

The structure of neuromelanin as studied by chemical degradative methods

The biosynthesis, structure and function of neuromelanin (NM), the dark brown melanin-like pigment present in the substantia nigra (SN), are not well characterized, in spite of the possible involvement of NM in the etiology and pathogenesis of Parkinson's disease. NM was isolated from the SN of five non-Parkinsonian human brains. NM and synthetic melanins, employed as models, were characterized by chemical analysis. Alkaline hydrogen peroxide (H2O2) oxidation of NM generated four degradation products, pyrrole-2,3-dicarboxylic acid (PDCA), pyrrole-2,3,5-tricarboxylic acid (PTCA), thiazole-4,5-dicarboxylic acid (TDCA) and thiazole-2,3,5-tricarboxylic acid (TTCA), whose ratios, especially the TTCA to PDCA ratio, indicate that NM is derived mostly from dopamine (DA) with 25% incorporation of cysteine (Cys) in the form of a benzothiazine structure. Hydriodic acid (HI) reductive hydrolysis of NM yielded 4-amino-3-hydroxyphenylethylamine (4-AHPEA) as a marker of cysteinyldopamine (CysDA)-derived units. The 4-AHPEA to PDCA ratio indicates a 21% incorporation of CysDA-derived units into NM. These degradative experiments also suggest that DOPA is not incorporated into NM to a significant extent (approximately 6% the level of DA). It is concluded that the TTCA to PDCA ratio is a useful indicator of CysDA-derived units in NM, and NM consists mainly of DA-melanin with some contribution from CysDA-melanin. The involvement of DA and CysDA as building blocks of NM demonstrates the detoxifying role of NM synthesis, since it prevents the intraneuronal accumulation of DA and CysDA, which would cause toxic effects.     

Alpha‐Synuclein as a Pathological Link Between Chronic Traumatic Brain Injury and Parkinson's Disease

The long‐term consequences of traumatic brain injury (TBI) are closely associated with the development of histopathological deficits. Notably, TBI may predispose long‐term survivors to age‐related neurodegenerative diseases, such as Parkinson's disease (PD), which is characterized by a gradual degeneration of the nigrostriatal dopaminergic neurons. However, preclinical studies on the pathophysiological changes in substantia nigra (SN) after chronic TBI are lacking. In the present in vivo study, we examined the pathological link between PD‐associated dopaminergic neuronal loss and chronic TBI. Sixty days post‐TBI, rats were euthanized and brain tissues harvested. Immunostaining was performed using tyrosine hydroxylase (TH), an enzyme required for the synthesis of dopamine in neurons, α‐synuclein, a presynaptic protein that plays a role in synaptic vesicle recycling, and major histocompatibility complex II (MHCII), a protein found in antigen presenting cells such as inflammatory microglia cells, all key players in PD pathology. Unbiased stereology analyses revealed significant decrease of TH‐positive expression in the surviving dopaminergic neurons of the SN pars compacta (SNpc) relative to sham control. In parallel, increased α‐synuclein accumulation was detected in the ipsilateral SN compared to the contralateral SN in TBI animals or sham control. In addition, exacerbation of MHCII+ cells was recognized in the SN and cerebral peduncle ipsilateral to injury relative to contralateral side and sham control. These results suggest α‐synuclein as a pathological link between chronic effects of TBI and PD symptoms as evidenced by significant overexpression and abnormal accumulation of α‐synuclein in inflammation‐infiltrated SN of rats exposed to chronic TBI. J. Cell. Physiol. 230: 1024–1032, 2015. © 2014 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.     

Mitochondrial dysfunction in Parkinson's disease

Parkinson's disease (PD) is one of the most common neurodegenerative disorders characterized by resting tremor, rigidity, and bradykinesia. The primary cause of PD is still unknown, but oxidative stress and mitochondrial dysfunction have been implicated as important contributors to neuronal death in substantia nigra (SN) of PD. Considering neurons as post-mitotic cells, neurons could have error-avoiding mechanism against oxidative DNA damage. Indeed, several DNA repairing enzymes such as MTH1, OGG1, and MUTYH express in human brain. All the three enzymes up-regulated in the SN of PD patients, suggesting these three enzymes cooperate in mitochondrial DNA repairing in PD brain.     

Q-band EPR investigations of neuromelanin in control and Parkinson's disease patients

New insights into the understanding of the changes induced in the iron domain of neuromelanin (NM) upon development of Parkinson's disease (PD) have been gained by electron paramagnetic spectroscopy (EPR). The results of this study are compared with a previously reported variable temperature analysis of X-band EPR spectra of a NM specimen obtained from control brain tissues. The availability of high sensitivity instruments operating in the Q-band (34.4 GHz) allows us to deal with the low amounts of NM available from PD brains. The organization of iron in NM is in the form of polynuclear superparamagnetic/antiferromagnetic aggregates, but the lack of one or more signals in the EPR spectra of NM from PD suggests that the development of the pathology causes NM to decrease its ability to bind iron. Furthermore, the detection of the Mn(II) signal in the Q-band spectra is exploited as an additional internal probe to assess minor structural differences in iron domains of PD and control NM specimens.     

The solubility of alpha-synuclein in multiple system atrophy differs from that of dementia with Lewy bodies and Parkinson's disease

Intracellular inclusions containing alpha-synuclein (alpha SN) are pathognomonic features of several neurodegenerative disorders. Inclusions occur in oligodendrocytes in multiple system atrophy (MSA) and in neurons in dementia with Lewy bodies (DLB) and Parkinson's disease (PD). In order to identify disease-associated changes of alpha SN, this study compared the levels, solubility and molecular weight species of alpha SN in brain homogenates from MSA, DLB, PD and normal aged controls. In DLB and PD, substantial amounts of detergent-soluble and detergent-insoluble alpha SN were detected compared with controls in grey matter homogenate. Compared with controls, MSA cases had significantly higher levels of alpha SN in the detergent-soluble fraction of brain samples from pons and white matter but detergent-insoluble alpha SN was not detected. There was an inverse correlation between buffered saline-soluble and detergent-soluble levels of alpha SN in individual MSA cases suggesting a transition towards insolubility in disease. The differences in solubility of alpha SN between grey and white matter in disease may result from different processing of alpha SN in neurons compared with oligodendrocytes. Highly insoluble alpha SN is not involved in the pathogenesis of MSA. It is therefore possible that buffered saline-soluble or detergent-soluble forms of alpha SN are involved in the pathogenesis of other alpha SN-related diseases.     

Neuropathology in mice expressing mouse alpha-synuclein

α-Synuclein (αSN) in human is tightly linked both neuropathologically and genetically to Parkinson's disease (PD) and related disorders. Disease-causing properties in vivo of the wildtype mouse ortholog (mαSN), which carries a threonine at position 53 like the A53T human mutant version that is genetically linked to PD, were never reported. To this end we generated mouse lines that express mαSN in central neurons at levels reaching up to six-fold compared to endogenous mαSN. Unlike transgenic mice expressing human wildtype or mutant forms of αSN, these mαSN transgenic mice showed pronounced ubiquitin immunopathology in spinal cord and brainstem. Isoelectric separation of mαSN species revealed multiple isoforms including two Ser129-phosphorylated species in the most severely affected brain regions. Neuronal Ser129-phosphorylated αSN occurred in granular and small fibrillar aggregates and pathological staining patterns in neurites occasionally revealed a striking ladder of small alternating segments staining either for Ser129-phosphorylated αSN or ubiquitin but not both. Axonal degeneration in long white matter tracts of the spinal cord, with breakdown of myelin sheaths and degeneration of neuromuscular junctions with loss of integrity of the presynaptic neurofilament network in mαSN transgenic mice, was similar to what we have reported for mice expressing human αSN wildtype or mutant forms. In hippocampal neurons, the mαSN protein accumulated and was phosphorylated but these neurons showed no ubiquitin immunopathology. In contrast to the early-onset motor abnormalities and muscle weakness observed in mice expressing human αSN, mαSN transgenic mice displayed only end-stage phenotypic alterations that manifested alongside with neuropathology. Altogether these findings show that increased levels of wildtype mαSN does not induce early-onset behavior changes, but drives end-stage pathophysiological changes in murine neurons that are strikingly similar to those evoked by expression of human wildtype or mutant forms.     

Tyrosinase exacerbates dopamine toxicity but is not genetically associated with Parkinson's disease

Tyrosinase is a key enzyme in the synthesis of melanin in skin and hair and has also been proposed to contribute to the formation of neuromelanin (NM). The presence of NM, which is biochemically similar to melanin in peripheral tissues, identifies groups of neurons susceptible in Parkinson's disease (PD). Whether tyrosinase is beneficial or detrimental to neurons is unclear; whilst the enzyme activity of tyrosinase generates dopamine-quinones and other oxidizing compounds, NM may form a sink for such radical species. In the present study, we demonstrated that tyrosinase is expressed at low levels in the human brain. We found that mRNA, protein and enzyme activity are all present but at barely detectable levels. In cell culture systems, expression of tyrosinase increases neuronal susceptibility to oxidizing conditions, including dopamine itself. We related these in vitro observations to the human disease by assessing whether there was any genetic association between the gene encoding tyrosinase and idiopathic PD. We found neither genotypic or haplotypic association with three polymorphic markers of the gene. This argues against a strong genetic association between tyrosinase and PD, although the observed contribution to cellular toxicity suggests that a biochemical association is likely.     

Do mtDNA Mutations Participate in the Pathogenesis of Sporadic Parkinson’s Disease?

The pathogenesis of sporadic Parkinson’s disease (PD) remains enigmatic. Mitochondrial complex-I defects are known to occur in the substantia nigra (SN) of PD patients and are also debated in some extracerebral tissues. Early sequencing efforts of the mitochondrial DNA (mtDNA) did not reveal specific mutations, but a long lasting discussion was devoted to the issue of randomly distributed low level point mutations, caused by oxidative stress. However, a potential functional impact remained a matter of speculation, since heteroplasmy (mutational load) at any base position analyzed, remained far below the relevant functional threshold. A clearly age-dependent increase of the ‘common mtDNA deletion’ had been demonstrated in most brain regions by several authors since 1992. However, heteroplasmy did hardly exceed 1% of total mtDNA. It became necessary to exploit PCR techniques, which were able to detect any deletion in a few microdissected dopaminergic neurons of the SN. In 2006, two groups published biochemically relevant loads of somatic mtDNA deletions in these neurons. They seem to accumulate to relevant levels in the SN dopaminergic neurons of aged individuals in general, but faster in those developing PD. It is reasonable to assume that this accumulation causes mitochondrial dysfunction of the SN, although it cannot be taken as a final proof for an early pathogenetic role of this dysfunction. Recent studies demonstrate a distribution of deletion breakpoints, which does not differ between PD, aging and classical mitochondrial disorders, suggesting a common, but yet unknown mechanism.     

Increased Oxidative Damage and Decreased Antioxidant Function in Aging Human Substantia Nigra Compared to Striatum: Implications for Parkinson’s Disease

Parkinson’s disease (PD) is characterized by selective degeneration and loss of dopaminergic neurons in the substantia nigra (SN) of the ventral mid brain leading to dopamine depletion in the striatum. Oxidative stress and mitochondrial damage have been implicated in the death of SN neurons during the evolution of PD. In our previous study on human PD brains, we observed that compared to SN, striatum was significantly protected against oxidative damage and mitochondrial dysfunction. To understand whether brain aging contributes to the vulnerability of midbrain to neurodegeneration in PD compared to striatum, we assessed the status of oxidant and antioxidant markers, glutathione metabolic enzymes, glial fibrillary acidic protein (GFAP) expression and mitochondrial complex I(CI) activity in SN (n = 23) and caudate nucleus (n = 24) during physiological aging in human brains. We observed a significant increase in protein oxidation (P < 0.001), loss of CI activity (P = 0.04) and increased astrocytic proliferation indicated by GFAP expression (P < 0.001) in SN compared to CD with increasing age. These changes were attributed to significant decrease in antioxidant function represented by superoxide dismutase (SOD) (P = 0.03), glutathione (GSH) peroxidase (GPx) (P = 0.02) and GSH reductase (GR) (P = 0.03) and a decreasing trend in total GSH and catalase with increasing age. However, these parameters were relatively unaltered in CD. We propose that SN undergoes extensive oxidative damage, loss of antioxidant and mitochondrial function and increased GFAP expression during physiological aging which might make it more vulnerable to neurotoxic insults thus contributing to selective degeneration during evolution of PD.     

Substantia nigra degeneration and tyrosine hydroxylase depletion caused by excess S-adenosylmethionine in the rat brain

The major symptoms of Parkinson's disease (PD) are tremors, hypokinesia, rigidity, and abnormal posture, caused by degeneration of dopamine (DA) neurons in the substantia nigra (SN) and deficiency of DA in the neostriatal dopaminergic terminals. Norepinephrine, serotonin, and melanin pigments are also decreased and cholinergic activity is increased. The cause of PD is unknown. Increased methylation reactions may play a role in the etiology of PD, because it has been observed recently that the CNS administration of S-adenosyl-l-methionine (SAM), the methyl donor, caused tremors, hypokinesia, and rigidity; symptoms that resemble those that occur in PD. Furthermore, many of the biochemical changes seen in PD resemble changes that could occur if SAM-dependent methylation reactions are increased in the brain, and interestingly,l-DOPA, the most effective drug used to treat PD, reacts avidly with SAM. So methylation may be important in PD; an idea that is of particular interest because methylation reactions increase in aging, the symptoms of PD are strikingly similar to the neurological and functional changes seen in advanced aging, and PD is age-related. For methylation to be regarded as important in PD it means that, along with its biochemical reactions and behavioral effects, increased methylation should also cause specific neuronal degeneration. To know this, the effects of an increase in methylation in the brain were studied by injecting SAM into the lateral ventricle of rats. The injection of SAM caused neuronal degeneration, noted by a loss of neurons, gliosis, and increased silver reactive fibers in the SN. The degeneration was accompanied with a decrease in SN tyrosine hydroxylase (TH) immunoreactivity, and degeneration of TH-containing fibers. At the injection site in the lateral ventricle it appears that SAM caused a weakening or dissolution of the intercellular substances; observed as a disruption of the ependymal cell layer and the adjacent caudate tissues. SAM may also cause brain atrophy; evidenced by the dilation of the cerebral ventricle. Most of the SAM-induced anatomical changes that were observed in the rat model are similar to the changes that occur in PD, which further support a role of SAM-dependent increased methylation in PD.     

Plasma Ceruloplasmin Ferroxidase Activity Correlates with the Nigral Sonographic Area in Parkinson’s Disease Patients: A Pilot Study

Increased area of the substantia nigra (SN) associated to iron deposition has been proposed as a specific marker for Parkinson’s disease (PD). Echogenicity, assessed by transcranial sonography (TCS), has been used to measure such an iron deposition. On the other hand, ferroxidase activity is known to play a role in brain iron metabolism and thus could be involved in increased SN echogenicity of PD patients. The present study was conducted to search for a possible correlation between both markers: TCS of SN and plasma ferroxidase activity. Twenty-one PD patients and 13 healthy volunteers (HV) were included. Mean SN sonographic areas were 0.31 cm² for PD patients and 0.12 cm² for HV (P < 0.001), while plasma ferroxidase activity was reduced in PD patients (P < 0.001). Interestingly, plasma ferroxidase activity was inversely correlated with the SN size by TCS (R² = 0.31), suggesting a relationship between the two markers.     

Genetic, biochemical, and characterization of neurological mutant 3, a new mouse model for Parkinson's disease

We have identified a new mutant mouse that we have named new mouse neurological mutant 3 (NM3); it may be a useful model to understand the underlying molecular and genetic basis of Parkinson's disease (PD). A mouse carrying the NM3 mutation arose spontaneously in an RIIIS/J breeding colony and was identified as having a movement disorder. Upon neurological examination of these mice, their movement was found to be slow and abnormal, with characteristic choreaform and bradykinetic-type movements, typical of PD. The importance of the gene mutation in NM3 in the molecular pathway involved in this pathology is underscored by the fact that these mice do not survive past weaning age if they are homozygous for the genetic mutation. We localized the gene mutation by positional cloning and genetic mapping to mouse chromosome 2 in an area that corresponds to human chromosome 2q24-31, which does not contain any known genes associated with PD. However, there was a significant decrease of 15-20% in the levels of dopamine, and its principal metabolite, 3,4-dihydroxyphenylacetic acid, in the midbrain of affected mice. Low concentrations of these substances are associated with PD in human patients, making these mutant mice candidates for studies of this disease.     

MAO-B elevation in mouse brain astrocytes results in Parkinson's pathology

Age-related increases in monoamine oxidase B (MAO-B) may contribute to neurodegeneration associated with Parkinson's disease (PD). The MAO-B inhibitor deprenyl, a long-standing antiparkinsonian therapy, is currently used clinically in concert with the dopamine precursor L-DOPA. Clinical studies suggesting that deprenyl treatment alone is not protective against PD associated mortality were targeted to symptomatic patients. However, dopamine loss is at least 60% by the time PD is symptomatically detectable, therefore lack of effect of MAO-B inhibition in these patients does not negate a role for MAO-B in pre-symptomatic dopaminergic loss. In order to directly evaluate the role of age-related elevations in astroglial MAO-B in the early initiation or progression of PD, we created genetically engineered transgenic mice in which MAO-B levels could be specifically induced within astroglia in adult animals. Elevated astrocytic MAO-B mimicking age related increase resulted in specific, selective and progressive loss of dopaminergic neurons in the substantia nigra (SN), the same subset of neurons primarily impacted in the human condition. This was accompanied by other PD-related alterations including selective decreases in mitochondrial complex I activity and increased mitochondrial oxidative stress. Along with a global astrogliosis, we observed local microglial activation within the SN. These pathologies correlated with decreased locomotor activity. Importantly, these events occurred even in the absence of the PD-inducing neurotoxin MPTP. Our data demonstrates that elevation of murine astrocytic MAO-B by itself can induce several phenotypes of PD, signifying that MAO-B could be directly involved in multiple aspects of disease neuropathology. Mechanistically this may involve increases in membrane permeant H(2)O(2) which can oxidize dopamine within dopaminergic neurons to dopaminochrome which, via interaction with mitochondrial complex I, can result in increased mitochondrial superoxide. Our inducible astrocytic MAO-B transgenic provides a novel model for exploring pathways involved in initiation and progression of several key features associated with PD pathology and for therapeutic drug testing.     

Meta-analysis of iron metabolism markers levels of Parkinson’s disease patients determined by fluid and MRI measurements

BackgroundParkinson’ s disease (PD) is a progressive neurodegenerative disease featured neuropathologically by the loss of dopaminergic neurons of the substantia nigra (SN). Iron overload in the SN is mainly relative to the pathology and pathogenesis of PD. Postmortem samples of PD has indicated the increased levels of brain iron. However, there is no consensus on iron content through iron-sensitive magnetic resonance imaging (MRI) techniques and the alteration of iron and iron related metabolism markers levels in blood and cerebrospinal fluids (CSF) are still unclear based on the current studies. In this study, we performed a meta-analysis to explore the iron concentration and iron metabolism markers levels through iron-sensitive MRI quantification and body fluid.MethodsA comprehensive literature search was performed in PubMed, EMBASE and Cochrane Library databases for relevant published studies that analyzed iron load in the SN of PD patients using quantitative susceptibility mapping (QSM) or susceptibility weighting imaging (SWI), and iron metabolism markers, iron, ferritin, transferrin, total iron-binding capacity（TIBC）in CSF sample or serum/plasma sample (from Jan 2010 to Sep 2022 to filter these inaccurate researches attributed to unadvanced equipment, inaccurate analytical methods). Standardized mean differences (SMD) or mean differences (MD) and 95% confidence intervals (CI) with random or fixed effect model was used to estimate the results.ResultsForty-two articles fulfilled the inclusion criteria including 19 for QSM, 6 for SWI, and 17 for serum/plasma/CSF sample including 2874 PD patients and 2821 healthy controls (HCs). Our meta-analysis results founded a notable difference for QSM values increase (19.67, 95% CI=18.69–20.64) and for SWI measurements (−1.99, 95% CI= −3.52 to −0.46) in the SN in PD patients. However, the serum/plasma/CSF iron levels and serum/plasma ferritin, transferrin, total iron-binding capacity (TIBC) did not differ significantly between PD patients and HCs.ConclusionsOur meta-analysis showed the consistent increase in the SN in PD patients using QSM and SWI techniques of iron-sensitive MRI measures while no significant differences were observed in other iron metabolism markers levels.     

PEP-1–ribosomal protein S3 protects dopaminergic neurons in an MPTP-induced Parkinson's disease mouse model

Parkinson's disease (PD) is a neurodegenerative disease characterized by a gradual loss of dopaminergic (DA) neurons in the substantia nigra (SN) of the brain. Ribosomal protein S3 (rpS3) has multiple functions related to protein synthesis, antioxidative activity, and UV endonuclease III activity. We have previously shown that PEP-1–rpS3 inhibits skin inflammation and provides neuroprotection against experimental cerebral ischemic damage. In this study, we examined whether PEP-1–rpS3 can protect DA neurons against oxidative stress in SH-SY5Y neuroblastoma cells and in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model. PEP-1–rpS3 was efficiently delivered to SH-SY5Y cells and the SN of the brain as confirmed by Western blot and immunohistochemical analysis. PEP-1–rpS3 significantly inhibited reactive oxygen species generation and DNA fragmentation induced by 1-methyl-4-phenylpyridinium, consequently leading to the survival of SH-SY5Y cells. The neuroprotection was related to the antiapoptotic activity of PEP-1–rpS3 that affected the levels of proapoptotic and antiapoptotic mediators. In addition, immunohistochemical data collected using a tyrosine hydroxylase antibody and cresyl violet staining demonstrated that PEP-1–rpS3 markedly protected DA cells in the SN against MPTP-induced oxidative stress. Therefore, our results suggest that PEP-1–rpS3 may be a potential therapy for PD.     

Identification of Synaptosomal Proteins Binding to Monomeric and Oligomeric α-Synuclein

Monomeric α-synuclein (αSN) species are abundant in nerve terminals where they are hypothesized to play a physiological role related to synaptic vesicle turn-over. In Parkinson’s disease (PD) and dementia with Lewy body (DLB), αSN accumulates as aggregated soluble oligomers in terminals, axons and the somatodendritic compartment and insoluble filaments in Lewy inclusions and Lewy neurites. The autosomal dominant heritability associated to mutations in the αSN gene suggest a gain of function associated to aggregated αSN. We have conducted a proteomic screen to identify the αSN interactome in brain synaptosomes. Porcine brain synaptosomes were fractionated, solubilized in non-denaturing detergent and subjected to co-immunoprecipitation using purified recombinant human αSN monomers or oligomers as bait. The isolated αSN binding proteins were identified with LC-LTQ-orbitrap tandem mass spectrometry and quantified by peak area using Windows client application, Skyline Targeted Proteomic Environment. Data are available via ProteomeXchange with identifier PXD001462. To quantify the preferential binding an average fold increase was calculated by comparing binding to monomer and oligomer. We identified 10 proteins preferentially binding monomer, and 76 binding preferentially to oligomer and a group of 92 proteins not displaying any preferred conformation of αSN. The proteomic data were validated by immunoprecipitation in both human and porcine brain extracts using antibodies against monomer αSN interactors: Abl interactor 1, and myelin proteolipid protein, and oligomer interactors: glutamate decarboxylase 2, synapsin 1, glial fibrillary acidic protein, and VAMP-2. We demonstrate the existence of αSN conformation selective ligands and present lists of proteins, whose identity and functions will be useful for modeling normal and pathological αSN dependent processes.