Cheek cell–derived α-synuclein and DJ-1 do not differentiate Parkinson's disease from control

Recently, α-synuclein (α-syn) and DJ-1, 2 proteins critically involved in Parkinson's disease (PD), have been shown to be present in saliva, suggesting their potential utility as biomarkers of PD. However, the origin and influence of demographic characteristics (e.g., age or sex) on these proteins are unknown. We identified cheek epithelium, which forms the majority of the cellular component of saliva and is readily accessible clinically, as 1 of several potential sources of salivary α-syn and DJ-1. However, no PD-related trend in the cellular component was present. In the supernatant collected from 198 healthy subjects, no correlation was seen between salivary DJ-1 or α-syn with age. When male and female subjects were analyzed separately, a weak age-dependent increase in DJ-1 level was present in male subjects, along with slightly increased α-syn in female subjects. These results, albeit largely negative, provide critical information for understanding the salivary gland pathology and saliva as a PD biomarker source, and must be considered in future investigations of salivary changes in PD.     

Plasma levels of DJ-1 as a possible marker for progression of sporadic Parkinson's disease

DJ-1 is a multifunctional protein whose loss of function by gene mutations may play a causative role for familial Parkinson's disease (PD). A recent study has shown that the expression of this molecule is upregulated in both brains and cerebrospinal fluids (CSF) in various neurological disorders, including sporadic PD, Alzheimer's disease (AD) and stroke, raising a possibility that DJ-1 could be a potential biomarker for these diseases. In this context, the main objective of the present study was to determine if DJ-1 was increased in the plasma of PD patients. For this purpose, blood plasma samples collected from sporadic PD patients, dementia with Lewy bodies (DLB) and healthy age-matched controls were analyzed by immunoblotting and enzyme-linked immunosorbent assay. The results showed that the plasma DJ-1 levels in PD (n = 104) were higher than those in control (n = 80) (p < 0.05). Moreover, the plasma DJ-1 levels in the advanced stage of PD (n = 52, Yahr III–IV) were higher than those in the early stage of PD (n = 52, Yahr I–II) (p < 0.05), demonstrating that the plasma DJ-1 was correlated with the disease severity in PD. Plasma DJ-1 levels were also significantly higher in DLB (n = 30) compared with both controls and early stage of PD (p < 0.01). Taken together, these results suggest that the plasma DJ-1 could be a useful biomarker for the evaluation of the disease severity in PD and possibly in other Lewy body diseases.     

Preparation and application of monoclonal antibodies against oxidized DJ-1. Significant elevation of oxidized DJ-1 in erythrocytes of early-stage Parkinson disease patients

DJ-1 was initially identified as a novel oncogene and has recently been found to be a causative gene for a familial form of Parkinson's disease (PD), viz, PARK7. Cysteine residue at position 106 (Cys-106) in DJ-1 was found to be oxidized preferentially under oxidative stress. In the present study, we developed specific antibodies against Cys-106-oxidized DJ-1 using baculovirus particles displaying the surface glycoprotein gp64-fusion protein as the immunizing agent. Western blot analysis combined with two-dimensional gel electrophoresis revealed that these antibodies specifically recognized oxidized DJ-1. Furthermore, we developed a competitive enzyme-linked immunosorbent assay (ELISA) for detecting oxidized DJ-1 and measured blood levels of oxidized DJ-1 in PD patients (n = 15). It was observed that the levels of oxidized DJ-1 in erythrocytes of unmedicated PD patients were markedly higher without overlap than those of medicated PD patients and healthy subjects. No significant difference was observed in DJ-1 levels between mediated and unmediated PD patient. These results suggest the oxidative modification of DJ-1 in PD patients and the potential application of the antibody for diagnosis of PD at early-stage.     

Neuroprotective effects of mesenchymal stem cells through autophagy modulation in a parkinsonian model

Autophagy is a major degradation pathway for abnormal aggregated proteins and organelles that cause various neurodegenerative diseases. Current evidence suggests a central role for autophagy in pathogenesis of Parkinson's disease, and that dysfunction in the autophagic system may lead to α-synuclein accumulation. In the present study, we investigated whether mesenchymal stem cells (MSCs) would enhance autophagy and thus exert a neuroprotective effect through the modulation of α-synuclein in parkinsonian models. In MPP⁺-treated neuronal cells, coculture with MSCs increased cellular viability, attenuated expression of α-synuclein, and enhanced the number of LC3-II-positive autophagosomes compared with cells treated with MPP⁺ only. In an MPTP-treated animal model of Parkinson's disease, MSC administration significantly increased final maturation of late autophagic vacuoles, fusion with lysosomes. Moreover, MSC administration significantly reduced the level of α-synuclein in dopaminergic neurons, which was elevated in MPTP-treated mice. These results suggest that MSC treatment significantly enhances autophagolysosome formation and may modulate α-synuclein expression in parkinsonian models, which may lead to increased neuronal survival in the presence of neurotoxins.     

Alpha‐Synuclein as a Biomarker for Parkinson's Disease

Parkinson's disease (PD) is a common neurodegenerative disorder, characterized pathologically by the presence of α‐synuclein (α‐syn)‐rich Lewy bodies. As clinical diagnosis of PD is challenging, misdiagnosis is common, highlighting the need for disease‐specific and early stage biomarkers. Both early diagnosis of PD and adequate tracking of disease progression could significantly improve outcomes for patients, particularly in regard to existing and future disease modifying treatments. Given its critical roles in PD pathogenesis, α‐syn may be useful as a biomarker of PD. The aim of this review is, therefore, to summarize the efficacy of tissue and body fluid α‐syn measurements in the detection of PD as well as monitoring disease progression. In comparison to solid tissue specimens and biopsies, biofluid α‐syn levels may be the most promising candidates in PD diagnosis and progression based on specificity, sensitivity and availability. Although α‐syn has been tested most extensively in cerebrospinal fluid (CSF), the relatively invasive procedure for collecting CSF is not suitable in most clinical settings, leading to investigation of plasma, blood and saliva as alternatives. The exploration of combined biomarkers, along with α‐syn, to improve diagnostic accuracy is also likely required.     

DJ-1 and αSYN in LRRK2 CSF do not correlate with striatal dopaminergic function

Previous studies demonstrated decreased levels of DJ-1 and α-synuclein (αSYN) in human cerebrospinal fluid (CSF) in patients with Parkinson's disease (PD), but neither marker correlated with PD severity, raising the possibility that they may be excellent progression markers during early or preclinical phases of PD. Individuals carrying the leucine-rich repeat kinase 2 (LRRK2) gene mutation are at increased risk for PD, and the phenotype of LRRK2 patients is almost identical to sporadic PD. To determine whether dopaminergic dysfunction in the basal ganglia, as determined by positron emission tomography (PET) scans, correlates with CSF levels of DJ-1 and αSYN during preclinical stages, Luminex assays were used to analyze CSF samples from asymptomatic LRRK2 mutation carriers, along with carriers who presented with a clinical diagnosis of PD. The data revealed no statistically significant relationship between PET scan evidence of loss of striatal dopaminergic function and the CSF biomarkers DJ-1 and αSYN, except for a weak correlation between DJ-1 and methylphenidate binding, suggesting that the use of these potential biomarkers on their own to screen LRRK2 gene mutation carriers for PD is not appropriate.     

Autophagy protects the rotenone-induced cell death in α-synuclein overexpressing SH-SY5Y cells

Loss of dopaminergic cells induced by α-synuclein accumulation in substantia nigra causes the development of Parkinson's disease (PD). To date, although autophagy has been implicated in the pathology of PD, the molecular mechanism is still unclear. To study the role of autophagy in PD pathogenesis, we established stable SH-SY5Y cell lines overexpressing wild-type or mutant α-synuclein proteins (A30P or A53T). Overexpression of mutant α-synuclein induced some protein aggregates and cell death in the absence of drug. LC3-II protein, a critical marker for autophagy, was produced in an autophagy-dependent manner. The rotenone-induced cell death was interrupted by autophagy stimulation. Autophagy activation also restored the mitochondrial membrane potential (MMP) impaired by rotenone in mutant α-synuclein expressing cells. Additionally, autophagy activation significantly relieved rotenone-induced ROS accumulation and HIF-1α expression in neuronal cells expressing mutant α-synuclein proteins. These findings indicate that autophagy plays an important scavenger role against harmful influence of toxic protein aggregates produced in rotenone-treated cells.     

DJ-1 mRNA anatomical localization and cell type identification in the mouse brain

Mutations in DJ-1 cause familial Parkinson's disease (PD). The expression pattern of DJ-1 in the brain remains controversial. In the present study, we used DJ-1 deficient mice as negative controls and examined DJ-1 mRNA expression in mouse brains. In sequential double labeling on the same sections, in situ hybridization of DJ-1 mRNA was followed by immunofluorescence detection of cell type markers. We found that DJ-1 mRNA was expressed in the majority of neurons in all brain areas examined. In particular, all dopamine neurons in the ventral midbrain expressed DJ-1 mRNA. In contrast, the choroid plexus and ependymal cells lining the ventricles were the only non-neuronal regions strongly expressing DJ-1 mRNA. DJ-1 mRNA was not detected in astrocytes. The fact that DJ-1 mRNA is expressed in all nigra dopamine neurons but not in astrocytes suggests that its potential neuroprotective role could be cell-autonomous. Moreover, that DJ-1 expression is not restricted to substantia nigra dopamine neurons suggests that PD-linked mutant DJ-1 may interact with other predisposing factors to cause the relatively selective dopamine neuron degeneration in Parkinson's disease.     

Synphilin-1 exhibits trophic and protective effects against Rotenone toxicity

Synphilin-1 is a cytoplasmic protein with unclear function. Synphilin-1 has been identified as an interaction partner of α-synuclein. The interaction between synphilin-1 and α-synuclein has implications in Parkinson's disease. In this study, we stably overexpressed human synphilin-1 in mouse N1E-115 neuroblastoma cells. We found that overexpression of synphilin-1 shortened cell growth doubling time and increased neurite outgrowth. Knockdown of endogenous synphilin-1 caused neuronal toxicity and shortened neurite outgrowth. We further found that synphilin-1 increased activation of the extracellular signal-regulated kinases (ERK1/2) and mediated neurite outgrowth. Rotenone, mitochondrial complex I inhibitor, has been shown previously to induce dopaminergic neurodegeneration and Parkinsonism in rats and Drosophila. We found that Rotenone induced apoptotic cell death in N1E-115 cells via caspase-3 activation and poly (ADP-ribose) polymerase (PARP) cleavage. Overexpression of synphilin-1 significantly reduced Rotenone-induced cell death, caspase-3 activation and PARP cleavage. The results indicate that synphilin-1 displays trophic and protective effects in vitro, suggesting that synphilin-1 may play a protective role in Parkinson's disease (PD) pathogenesis and may lead to a potential therapeutic target for PD intervention.     

Regional differences in the severity of Lewy body pathology across the olfactory cortex

We studied α-synuclein pathology in the rhinencephalon of ten cases of Parkinson's disease (PD) and twelve neurologically normal controls, of which seven had incidental Lewy bodies in the substantia nigra at autopsy and five had no pathological evidence of neurological disease. In all PD and incidental Lewy bodies cases, α-synuclein pathology was found in all five subregions of the primary olfactory cortex that were sampled, and amongst them the pathology was significantly more severe in the temporal division of the piriform cortex than in the frontal division of the piriform cortex, olfactory tubercle or anterior portions of the entorhinal cortex. The orbitofrontal cortex, which is an area of projection from the primary olfactory cortex, was affected in some cases but overall the α-synuclein pathology was less severe in this area than in the primary olfactory cortex. Because different areas of the rhinencephalon are likely to play different roles in olfaction and our data indicate a differential involvement by α-synuclein deposition of structures implicated in smell, future prospective studies investigating the pathophysiological basis of hyposmia in PD should consider to examine the areas of primary olfactory cortex separately.     

Clinical availability of skin biopsy in the diagnosis of Parkinson's disease

To determine whether skin biopsy is practically useful in the premortem diagnosis for Parkinson's disease (PD), we examined Lewy pathology in the skin of the chest wall and leg, obtained from 6-mm punch biopsies, using phosphorylated α-synuclein antibody in 20 patients with clinically diagnosed PD. Abnormal accumulation of α-synuclein was found in the chest skin of two (10%) of 20 patients, but not in the leg. Although skin biopsy combined with a conventional immunohistochemistry for α-synuclein is not sufficient as a diagnostic tool, we could firstly demonstrate Lewy pathology in premortem tissue. The skin remains to be a promising tissue to be examined for the premortem diagnosis of PD.     

Cerebrospinal fluid from patients with multiple system atrophy promotes in vitro α-synuclein fibril formation

The aggregation of α-synuclein (αS) in the central nervous system (CNS) is the hallmark of multiple system atrophy (MSA) and Lewy body diseases including Parkinson's disease (PD) and dementia with Lewy bodies (DLB) (α-synucleinopathies). To test the hypothesis that patients with α-synucleinopathies have a CNS environment favorable for αS aggregation, we examined the influence of cerebrospinal fluid (CSF) from patients with MSA (n = 20), DLB (n = 8), and PD (n = 10) on in vitro αS fibril (fαS) formation at pH 7.5 and 37 °C using fluorescence spectroscopy with thioflavin S, compared with those with hereditary spinocerebellar ataxia (hSCA) (n = 16), and tension-type headache (n = 7). CSF from MSA patients (MSA-CSF) promoted fαS formation more strongly than PD-, hSCA-, or headache-CSF. By electron microscopic analyses, the width of fαS formed in MSA-CSF was significantly greater than others. MSA may have a CSF environment particularly favorable for fαS formation.     

Melatonin inhibits amphetamine-induced increase in α-synuclein and decrease in phosphorylated tyrosine hydroxylase in SK–N–SH cells

α-Synuclein is an abundant presynaptic protein implicated in neuronal plasticity and neurodegeneration disorders. Understanding α-synuclein function in dopaminergic cells could add to our knowledge of this key protein which is implicated in Parkinson's disease. Chronic or intermittent amphetamine (AMPH) abuse may create temporary or permanent disturbances in the dopaminergic system of the brain that may predispose individuals to Parkinsonism. Our previous studies showed that neurotoxicity induced by AMPH was mediated by enhanced oxidative stress and these effects were abolished by melatonin, a main secretory product of pineal gland. The present study was conducted to investigate the effect of AMPH on α-synuclein in regulating tyrosine hydroxylase (TH), a rate limiting enzyme for dopamine synthesis, in cultured human dopaminergic SK–N–SH cells. Of these, phosphorylation of Ser40 (pSer40) contributes significantly to TH activation and dopamine synthesis. Our data indicated that AMPH significantly increased the level of α-synuclein to 183% of the control value while reducing the levels of phosphorylated TH (TH-pSer40) enzyme and mitochondrial complex I to 78 and 52.9% of the control values, respectively and these effects were attenuated by melatonin. Further studies are needed to explore the mechanism by which α-synuclein contributes to TH-pSer40 dephosphorylation and the mechanism by which melatonin contributes to this interaction.     

Differential effects of overexpression of wild-type and mutant human α-synuclein on MPP-induced neurotoxicity in PC12 cells

The genetic background and the pathogenesis of familial Parkinson's disease (PD) have not been fully elucidated. Two missense mutations in the α-synuclein gene, A30P and A53T, have been linked to familial PD. Increasing evidence suggests the involvement of α-synuclein, the dopamine transporter (DAT), and neurotoxins in the pathogenesis of PD, but their relationships to the death of nigral cells remains poorly understood. In the present study, we used the PC12 cell line, a well recognized model of the nigral cell, to investigate the effects of overexpression of wild-type (WT) and mutant human α-synuclein on MPP⁺-induced neurotoxicity. We found that overexpression of mutant α-synuclein enhanced the toxicity of MPP⁺ to PC12 cells and elevated intracellular reactive oxygen species (ROS) levels, whereas overexpression of WT α-synuclein protected PC12 cells against MPP⁺ toxicity and lowered ROS levels. Furthermore, assays of ¹³¹I-FP-β-CIT binding with DAT membrane fractions showed that WT and mutant α-synuclein had different effects on the expression of DAT on the cell membrane following exposure to MPP⁺. WT α-synuclein reduced the toxic effect of MPP⁺ by facilitating DAT internalization, while both A30P and A53T α-synuclein aggravated the toxic effect of MPP⁺ by reducing DAT internalization. These data indicate that α-synuclein regulates ROS levels and DAT surface expression in dopaminergic neurons, and thus changes the response of these cells to MPP⁺.     

Elevation of oxidized DJ-1 in the brain and erythrocytes of Parkinson disease model animals

DJ-1, the causative gene of a familial form of Parkinson's disease (PD), has been reported undergo oxidation preferentially at the 106th cysteine residue (Cys-106) under oxidative stress. Recently, it has been found that the levels of oxidized DJ-1 in erythrocytes of unmedicated PD patients are markedly higher than those in medicated PD patients and healthy subjects. In the present study, we examined the changes in oxidized DJ-1 levels in the brain and erythrocytes of PD animal models using specific antibodies against Cys-106-oxidized DJ-1. Treatment with PD model compounds such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 6-hydroxydopamine significantly elevated the levels of oxidized DJ-1 in erythrocytes. Immunohistochemical analysis also revealed that the number of oxidized DJ-1 antibody-positive cells in the substantia nigra of MPTP-treated mouse increased in a dose-dependent manner. These results suggest that the oxidative modification of DJ-1 in the brain and erythrocytes is involved in the pathogenesis of PD in animal models.     

Copper pathology in vulnerable brain regions in Parkinson's disease

Synchrotron-based x-ray fluorescence microscopy, immunofluorescence, and Western blotting were used to investigate changes in copper (Cu) and Cu-associated pathways in the vulnerable substantia nigra (SN) and locus coeruleus (LC) and in nondegenerating brain regions in cases of Parkinson's disease (PD) and appropriate healthy and disease controls. In PD and incidental Lewy body disease, levels of Cu and Cu transporter protein 1, were significantly reduced in surviving neurons in the SN and LC. Specific activity of the cuproprotein superoxide dismutase 1 was unchanged in the SN in PD but was enhanced in the parkinsonian anterior cingulate cortex, a region with α-synuclein pathology, normal Cu, and limited cell loss. These data suggest that regions affected by α-synuclein pathology may display enhanced vulnerability and cell loss if Cu-dependent protective mechanisms are compromised. Additional investigation of copper pathology in PD may identify novel targets for the development of protective therapies for this disorder.     

Dietary energy intake modifies brainstem autonomic dysfunction caused by mutant α-synuclein

Parkinson's disease (PD) patients often exhibit impaired regulation of heart rate by the autonomic nervous system (ANS) that may precede motor symptoms in many cases. Results of autopsy studies suggest that brainstem pathology, including the accumulation of α-synuclein, precedes damage to dopaminergic neurons in the substantia nigra in PD. However, the molecular and cellular mechanisms responsible for the early dysfunction of brainstem autonomic neurons are unknown. Here we report that mice expressing a mutant form of α-synuclein that causes familial PD exhibit aberrant autonomic control of the heart characterized by elevated resting heart rate and an impaired cardiovascular stress response, associated with reduced parasympathetic activity and accumulation of α-synuclein in the brainstem. These ANS abnormalities occur early in the disease process. Adverse effects of α-synuclein on the control of heart rate are exacerbated by a high energy diet and ameliorated by intermittent energy restriction. Our findings establish a mouse model of early dysregulation of brainstem control of the cardiovascular system in PD, and further suggest the potential for energy restriction to attenuate ANS dysfunction, particularly in overweight individuals.     

Fe and Cu do not differ in Parkinson's disease: A replication study plus meta-analysis

To evaluate whether iron and copper levels in serum, plasma, and cerebrospinal fluid are disarranged in Parkinson's disease (PD), we performed meta-analyses of 33 studies on the topic published from 1987 to 2011 and contextually carried out a replication study in serum by ourselves as well. We found no variation in metals between PD patients and healthy controls, according to our replication study. The metaregression for sex revealed that serum copper differences found in some studies could be referred to the different percentage of women in the PD sample. Transferrin and transferrin saturation levels found increased in PD subjects underline the concept to extend the iron study in PD to iron master proteins.