Molecular beacon-based detection of circulating microRNA-containing extracellular vesicle as an α-synucleinopathy biomarker.

Early and precise diagnosis of α-synucleinopathies is challenging but critical. In this study, we developed a molecular beacon-based assay to evaluate microRNA-containing extracellular vesicles (EVs) in plasma. We recruited 1203 participants including healthy controls (HCs) and patients with isolated REM sleep behavior disorder (iRBD), α-synucleinopathies, or non-α-synucleinopathies from eight centers across China. Plasma miR-44438-containing EV levels were significantly increased in α-synucleinopathies, including those in the prodromal stage (e.g., iRBD), compared to both non-α-synucleinopathy patients and HCs. However, there are no significant differences between Parkinson's disease (PD) and multiple system atrophy. The miR-44438-containing EV levels negatively correlated with age and the Hoehn and Yahr stage of PD patients, suggesting a potential association with disease progression. Furthermore, a longitudinal analysis over 16.3 months demonstrated a significant decline in miR-44438-containing EV levels in patients with PD. These results highlight the potential of plasma miR-44438-containing EV as a biomarker for early detection and progress monitoring of α-synucleinopathies.

Calcium influx: An essential process by which α-Synuclein regulates morphology of erythrocytes.

INTRODUCTION: Morphological abnormalities of erythrocytes/red blood cells (RBCs), e.g., increased acanthocytes, in Parkinson's disease (PD) have been reported previously, although the underlying mechanisms remain to be characterized. In this study, the potential roles of α-synuclein (α-syn), a protein critically involved in PD and highly abundant in RBCs, were studied in PD patients as well as in a PD mouse model. METHODS: Transgenic [PAC-Tg (SNCAA53T), A53T] mice overexpressing A53T mutant α-syn and SNCA knockout mice were employed to characterize the effect of α-syn on RBC morphology. In addition to A53T and SNCA knockout mice, the morphology of RBCs of PD patients was also examined using scanning electron microscopy. The potential roles of α-syn were further investigated in cultured RBCs and mice. RESULTS: Morphological abnormalities of RBCs and increased accumulation of aggregated α-syn on the RBC membrane were observed in PD patients. A similar phenomenon was also observed in A53T mice. Furthermore, while mice lacking α-syn expression showed a lower proportion of acanthocytes, treating RBCs derived from SNCA knockout mice with aggregated α-syn resulted in a higher percentage of acanthocytes. In a follow-up proteomic investigation, several major classes of proteins were identified as α-syn-associated proteins on the RBC membrane, seven of which were calcium-binding proteins. Applying aggregated α-syn to the RBC membrane directly induced extracellular calcium influx along with morphological changes; both observations were adequately reversed by blocking calcium influx. CONCLUSIONS: This study demonstrated that α-syn plays a critical role in PD-associated morphological abnormalities of RBCs, at least partially via a process mediated by extracellular calcium influx.

Erythrocytic alpha-synuclein as potential biomarker for the differentiation between essential tremor and Parkinson's disease.

Introduction: The differentiation between essential tremor (ET) and Parkinson's disease (PD) can be difficult because of the symptom overlaps. Erythrocytes are the major source of peripheral α-synuclein (α-syn), which is the most studied pathological molecular of PD. We have reported that erythrocytic α-syn levels in PD patients are significantly increased compared to those in healthy controls (HCs). However, little is known about the levels of erythrocytic α-syn species in ET patients. Methods: This study includes 15 patients with ET, 64 patients with PD, and 49 age and sex matched HCs. A well-established electrochemiluminescence assay was used to measure the erythrocytic total and aggregated α-syn levels. The receiver operating characteristic (ROC) curve analysis was applied to evaluate the diagnostic values of erythrocytic α-syn for ET diagnosis and differentiation. The correlations of erythrocytic α-syn levels with disease durations were tested using Spearman's Rank Correlation analysis. Results: We found that both erythrocytic total and aggregated α-syn concentrations are significantly increased in PD and ET patients compared to those in HCs. Erythrocytic total α-syn levels are significantly higher in ET patients than those in PD group. Furthermore, the ratios of erythrocytic aggregated to total α-syn levels in ET patients are significantly decreased than those in PD and HC subjects. We also found a significant association of erythrocytic aggregated α-syn levels with the disease duration of ET patients. Conclusion: Our findings suggest new insight into the changes of erythrocytic total and aggregated α-syn levels as potential biomarkers for ET patients.

Erythrocytic alpha-synuclein in early Parkinson's disease: A 3-year longitudinal study.

BACKGROUND: Early diagnosis of Parkinson's disease (PD) could significantly improve outcomes for patients and future disease-modifying treatments. Several studies have revealed that α-synuclein levels in peripheral erythrocytes are associated with PD, but the diagnostic value in early PD is still unknown. METHODS: This study included both cross-sectional and longitudinal design. The subjects included 45 patients with early PD and 79 age-matched healthy controls. Participants were re-examined with repeated blood collection and clinical assessments after 3 years. The electrochemiluminescence assay was used to measure total and oligomeric α-synuclein levels respectively. The diagnostic value of erythrocytic α-synuclein for early PD was determined by receiver operator characteristic (ROC) curve. Correlations between RBC α-synuclein levels and changes over 3 years in clinical characteristic scores were further investigated with a linear regression. RESULTS: Total and oligomeric α-synuclein levels in erythrocyte were significantly increased in early PD groups compared with control group (Total α-synuclein, p < 0.001; Oligomer, p < 0.001). Levels of total and oligomeric α-synuclein in erythrocytes were correlated with MDS-UPDRS III scores in early PD (Total α-synuclein, p = 0.008; Oligomer, p = 0.037). After adjusting for age, gender and dopaminergic medication, an association was found between higher erythrocytic oligomeric α-synuclein levels at baseline and greater increase in MDS-UPDRS III scores over 3 years (p = 0.007). CONCLUSION: Our study suggests that total and oligomeric α-synuclein in erythrocyte were elevated even in the initial motor stage of PD. Higher erythrocytic oligomeric α-synuclein levels at baseline predicts a faster clinical decline over time in patients with early PD.

Lower serum triglyceride levels linked to more severe motor performance in Parkinson's disease.

INTRODUCTION: Emerging evidence has suggested that lipid metabolism is correlated with Parkinson's disease (PD) onset and progression. However, the effect of lipid metabolism on motor performance in PD patients is still unknown. This study estimated the association between lipid profiles and the severity of motor performance in PD. METHODS: This cross-sectional study enrolled 279 idiopathic PD patients from the Department of Neurology of Beijing Tiantan Hospital from May 2016 to August 2018. Serum triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), apolipoprotein A1 (Apo-A1), and apolipoprotein B (Apo-B) levels were detected in fast serum samples. Motor performance was assessed by Movement Disorder Society-Unified Parkinson's Disease Rating Scale part III (MDS-UPDRS III) total scores and subscores in these patients. The associations of lipid profiles with motor performance were analyzed using multivariable linear regression models. RESULTS: Compared to males, females with PD exhibited significantly higher serum TC, LDL-C, HDL-C, Apo-A1, and Apo-B levels. When accounting for covariates, lower serum TG levels were significantly associated with higher MDS-UPDRS III total scores and gait/postural instability subscores. Additionally, the univariate linear regression model showed that in males with PD, serum HDL-C or Apo-A1 levels were significantly associated with tremor subscores. CONCLUSION: Lower serum TG levels were associated with more severe motor performance in patients with PD and TG may be a potential predictive biomarker for motor performance in PD patients.

Erythrocytic α-Synuclein Species for Parkinson's Disease Diagnosis and the Correlations With Clinical Characteristics.

BACKGROUND: Erythrocytes contain most of the peripheral α-synuclein (α-syn), which is the key pathological molecular of α-synucleinopathies including Parkinson's disease (PD). Our objectives were to assess the efficiency of erythrocytic total and oligomeric α-syn levels as PD diagnostic biomarkers, and to identify the correlations between erythrocytic α-syn levels and physiological/psychiatrical assessment scales. METHODS: Home-brewed electrochemiluminescence assays were applied to assess the concentrations of erythrocytic total and oligomeric α-syn levels in a cohort including 124 patients with PD and 79 healthy controls (HCs). The correlations between erythrocytic α-syn levels and clinical measurements were assessed using Spearman's rank test. RESULTS: Both the erythrocytic total and oligomeric α-syn levels were significantly higher in PD patients than HCs. The biomarkers adjusted for age and sex discriminated PDs from HCs well with 80% sensitivity, 89% specificity and 79% sensitivity, 83% specificity, respectively. Combining erythrocytic total and oligomeric α-syn levels by using binary logistic regression analysis with the controlling of age and sex generated a factor discriminates PDs from HCs with 88% sensitivity and 85% specificity. The erythrocytic total but not oligomeric α-syn levels adjusted for age and sex significantly correlated with anxiety scales and the MDS-UPDRS III scales in PD patients, respectively. CONCLUSION: We showed the usefulness of erythrocytic total and oligomeric α-syn levels as biomarkers for PD. Our results also suggest the capability of erythrocytic α-syn as a potential pathological factor and therapeutic target for psychiatric symptoms in PD patients.

Correlation Between Lacunae and the Wearing-off Phenomenon in Parkinson's Disease.

PURPOSE: Lacunae are imaging biomarkers of cerebral small vessel disease (CSVD) and are correlated with the degree of gait instability in Parkinson's disease (PD). The wearing-off phenomenon (WO) occurs more frequently in PD patients as disease progresses. The present study aimed to investigate the overall impact of the quantity and location of lacunae on the WO in PD. PATIENTS AND METHODS: This retrospective, single-center study included 315 consecutive eligible patients with PD from Beijing Tiantan Hospital from May 2016 to August 2018. We collected data on demographics and clinical features, assessed lacunae and examined the presence of the WO. The association between lacunae and the WO was assessed using a binary logistic regression model. RESULTS: The number of lacunae was significantly associated with the WO in patients with PD according to a model adjusted for age at onset, disease duration, Hoehn-Yahr (H-Y) staging, Movement Disorder Society-Unified Parkinson's Disease Rating Scale part III (MDS-UPDRS III) total score and levodopa equivalent daily dosage (LEED) (P=0.037, OR 1.156, 95% CI 1.009, 1.325) and to a model further adjusted for other CSVD imaging biomarkers (P=0.046, OR 1.172, 95% CI 1.003, 1.369). Following additional adjustment for other potential confounders, the association remained significant (P=0.043, OR 1.195, 95% CI 1.005, 1.421). Lacunae in subcortical areas (P=0.004, OR 0.498, 95% CI 0.308, 0.803) and basal ganglia (P=0.046, OR 1.616, 95% CI 1.009, 2.587), especially in the caudate nuclei (P=0.023, OR 1.104, 95% CI 0.185, 0.881), were significantly associated with the WO in PD patients. CONCLUSION: Our finding highlights the significant association between lacune and the WO, and lacunae may be an independent contributor to the WO in PD patients. Promoting neurovascular health may prevent the progression of the WO in PD patients.

Effect of onset age on the levodopa threshold dosage for dyskinesia in Parkinson's disease.

INTRODUCTION: With the levodopa threshold effect for dyskinesia observed, threshold dosage of levodopa was identified in the general Parkinson's disease (PD) population. While early-onset PD (EOPD) and late-onset PD (LOPD) differ in the pathogenesis and clinical manifestations, threshold dosage of levodopa for individualized treatment remains unestablished. The objective of this study was to propose threshold dosage of levodopa in EOPD and LOPD patients, respectively. METHODS: Data on demographic and clinical and treatment measures were collected in 539 PD patients. Patients were divided into different onset groups using 50 as the cut-off age. We used univariable and multivariable analysis to screen for risk factors for dyskinesia. Receiver operating characteristic curve was used to determine the levodopa threshold dosages for dyskinesia. RESULTS: The prevalence of dyskinesia was 47.7% (53/111) in the EOPD group and 24.1% (103/428) in the LOPD group. Risk factors identified for dyskinesia include high levodopa daily dose and levodopa responsiveness for EOPD patients and high levodopa daily dose, long levodopa treatment duration, low body weight, use of entacapone, and high Hoehn-Yahr stage in off state for LOPD patients. The daily levodopa threshold dosages were 400 mg or 5.9 mg/kg for EOPD and 450 mg or 7.2 mg/kg for LOPD. CONCLUSION: EOPD patients had lower levodopa threshold dosage comparing with LOPD patients. Treatment of EOPD requires stricter levodopa dose control to delay the onset of dyskinesia.

Reduced erythrocytic CHCHD2 mRNA is associated with brain pathology of Parkinson's disease.

Peripheral biomarkers indicative of brain pathology are critically needed for early detection of Parkinson's disease (PD). In this study, using NanoString and digital PCR technologies, we began by screening for alterations in genes associated with PD or atypical Parkinsonism in erythrocytes of PD patients, in which PD-related changes have been reported, and which contain ~ 99% of blood α-synuclein. Erythrocytic CHCHD2 mRNA was significantly reduced even at the early stages of the disease. A significant reduction in protein and/or mRNA expression of CHCHD2 was confirmed in PD brains collected at autopsy as well as in the brains of a PD animal model overexpressing α-synuclein, in addition to seeing a reduction of CHCHD2 in erythrocytes of the same animals. Overexpression of α-synuclein in cellular models of PD also resulted in reduced CHCHD2, via mechanisms likely involving altered subcellular localization of p300 histone acetyltransferase. Finally, the utility of reduced CHCHD2 mRNA as a biomarker for detecting PD, including early-stage PD, was validated in a larger cohort of 205 PD patients and 135 normal controls, with a receiver operating characteristic analysis demonstrating > 80% sensitivity and specificity.

Cerebral small vessel disease may worsen motor function, cognition, and mood in Parkinson's disease.

INTRODUCTION: Emerging evidence has suggested that cerebral small vessel disease (CSVD) may worsen motor function and cognition in Parkinson's disease (PD). However, the effect of CSVD on anxiety and depression in patients with PD remains unknown. This study explored the multi-dimensional effects of CSVD on PD outcomes (motor, cognition, and depression/anxiety). METHODS: This cross-sectional study included 431 patients with PD from Beijing Tiantan Hospital from May 2016 to August 2019. CSVD imaging markers were assessed and the four-point CSVD burden score was calculated. Motor function (MDS-UPDRS III score and subscores), cognition (MMSE, MoCA), anxiety (HAMA), and depression (HAMD) were assessed in these patients. The associations of CSVD with these outcomes were analyzed using the Spearman's correlation and multivariable linear regression models. RESULTS: Motor dysfunction, cognitive impairment, depression, and anxiety were significantly worse in patients with severe CSVD than in those with mild CSVD. Multivariable linear regression showed that CSVD burden was significantly associated with motor dysfunction (MDS-UPDRS III score and rigidity and bradykinesia subscores), impaired cognition, and high levels of depression and anxiety. A marginally significant association was observed between CSVD burden and gait/postural instability in multivariable regression analysis. Among the CSVD imaging markers, white matter hyperintensity, number of lacunes, and microbleeds were positively correlated with the severity of motor, cognitive, and emotional impairments, while the perivascular space in the basal ganglia was only correlated with cognitive impairments. CONCLUSIONS: Comorbid CSVD may affect multiple functional domains in patients with PD. Management of cerebrovascular disease may improve PD outcomes.

Clinical Characteristics and Electrophysiological Biomarkers of Parkinson's Disease Developed From Essential Tremor.

Background and Objective: Parkinson's disease developed from essential tremor (ET-PD) is a distinct clinical syndrome that is different from essential tremor (ET) and Parkinson's disease (PD). There is currently a lack of research on ET-PD. Tremor characteristics (amplitude and frequency) are primary quantitative indexes for diagnosing and monitoring of tremors. In this study, we aimed to explore specific clinical and electrophysiological biomarkers for the identification of ET-PD. Methods: The study included patients with ET-PD (n = 22), ET (n = 42), and tremor-dominant PD (t-PD, n = 47). We collected demographic data, clinical characteristics (including motor and non-motor symptoms), and tremor analysis. The frequency, amplitude, contracting patterns of resting tremor and postural tremor were collected. The analysis of ET-PD and ET/t-PD was compared. The receiver operating characteristic (ROC) curve was used to analyze the electrophysiological features in distinguishing ET-PD from ET or t-PD. Results: Compared with ET, hyposmia, bradykinesia, rigidity, postural abnormality, and resting tremor were more common in the ET-PD group (P = 0.01, 0.003, 0.001, 0.001, 0.019, respectively). The postural tremor frequencies of the head, upper limbs, and lower limbs were significantly lower in the ET-PD than in the ET (P = 0.007, 0.003, 0.035, respectively), which were the most appropriate variables for distinguishing ET-PD from ET (AUC: 0.775, 0.727, and 0.701, respectively). Compared with t-PD, bradykinesia, rigidity, postural abnormality (both P < 0.001), and resting tremor (P = 0.024) were less common in the ET-PD. The postural tremor amplitudes of the head and upper limbs were significantly higher in the ET-PD than in the t-PD (P = 0.022, 0.001, respectively), which were the most appropriate variables for distinguishing ET-PD from t-PD (AUC: 0.793 and 0.716). Conclusions: Hyposmia and electrophysiological biomarkers (postural tremor frequencies and amplitudes) help early recognition of ET-PD.

Differential Diagnosis of Multiple System Atrophy-Parkinsonism and Parkinson's Disease Using α-Synuclein and External Anal Sphincter Electromyography.

Background and aim: Discriminating multiple system atrophy-parkinsonism (MSA-P) from Parkinson's disease (PD) is challenging. We aimed to provide a new method to make an identification between MSA-P and PD by combining biofluid marker with electrophysiology marker. Methods: The XYCQ EV Enrichment KIT was applied to extract extracellular vesicles (EVs) from saliva. The levels of α-syn which included total α-syn (α- synTotal), phosphorylated-ser129 α-syn (α-synPS129) and oligomeric α-syn (α-synOlig) in EVs of saliva were tested by new developed Electrochemiluminescence (ECL) assays. We collected multi-motor unit potential (MUP) of all participants who conducted external anal sphincter electromyography (EAS-EMG). The duration, phase, amplitude and satellite potential of EAS-EMG were analyzed. The Receiver operator characteristic (ROC) curve was adopted to analyze the diagnostic utility of α-syn in EVs of saliva, EAS-EMG for MSA-P. Results: In EVs of saliva, the α-synTotal concentrations were lower in MSA-P than PD (P = 0.003). No significant difference was shown in α-synOlig and α-synPS129. α-synTotal 4.46 pg/ng distinguished MSA-P from PD with area under the curve (AUC) 0.804. Compared with PD, the duration, phase and satellite potential of EAS-EMG in MSA-P were increased (P = 0.002, 0.008, 0.001). There was no significant difference in amplitude. ROC curve showed that the duration (AUC: 0.780), phase (AUC: 0.751), and satellite potential (AUC: 0.809) had both diagnostic value for MSA-P. The combination of α-synTotal in salivary EVs and EAS-EMG (including duration, phase and satellite potential) could efficiently make a differentiation between MSA-P and PD with sensitivity of 100% and specificity of 86%. The AUC value was 0.901. Conclusion: The study suggested the combination of α-synTotal in salivary EVs and EAS-EMG could help efficiently distinguish MSA-P from PD.

Risk thresholds of levodopa dose for dyskinesia in Chinese patients with Parkinson's disease: a pilot study.

Levodopa is widely used to treat Parkinson's disease (PD), and its long-term therapy may induce dyskinesia in a dose-dependent manner. However, the threshold dose with a relatively low risk for dyskinesia has not been determined. Demographic, clinical profiles and detailed information of dopaminergic drugs were recorded for 403 PD patients in treatment with levodopa. Variables were compared between dyskinesia and non-dyskinesia groups. Logistic regression analysis was used to assess the association between levodopa dose-related variables and dyskinesia. Receiver operating characteristic curve and decision tree classification model were used to investigate the cut-off value of levodopa dose to best separate the dyskinesia group from the non-dyskinesia group. Patients with dyskinesia tended to have a lower weight and age at onset, higher percentage of female and wearing-off, longer duration of disease and levodopa treatment, higher H-Y stage and MDS-UPDRS Part III score, and higher levodopa dose and levodopa equivalent dose than those without dyskinesia. After adjusted for demographical and clinical variables, levodopa dose-related factors (daily dose, cumulative dose, and weight-adjusted dose) were still associated with dyskinesia. Both the receiver operating characteristic and decision tree classification analysis indicated that patients who have taken levodopa dose ≤ 400 mg per day may be associated with a reduced risk for dyskinesia. In conclusion, we evaluated the thresholds of levodopa treatment with a relatively low risk for dyskinesia. These data should be considered for prevention and management of dyskinesia in patients with PD.

Erythrocytic α-Synuclein as a potential biomarker for Parkinson's disease.

BACKGROUND: Erythrocytes are a major source of peripheral α-synuclein (α-Syn). The goal of the current investigation is to evaluate erythrocytic total, oligomeric/aggregated, and phosphorylated α-Syn species as biomarkers of Parkinson's disease (PD). PD and healthy control blood samples were collected along with extensive clinical history to determine whether total, phosphorylated, or aggregated α-Syn derived from erythrocytes (the major source of blood α-Syn) are more promising and consistent biomarkers for PD than are free α-Syn species in serum or plasma. METHODS: Using newly developed electrochemiluminescence assays, concentrations of erythrocytic total, aggregated and phosphorylated at Ser129 (pS129) α-Syn, separated into membrane and cytosolic components, were measured in 225 PD patients and 133 healthy controls and analyzed with extensive clinical measures. RESULTS: The total and aggregated α-Syn levels were significantly higher in the membrane fraction of PD patients compared to healthy controls, but without alterations in the cytosolic component. The pS129 level was remarkably higher in PD subjects than in controls in the cytosolic fraction, and to a lesser extent, higher in the membrane fraction. Combining age, erythrocytic membrane aggregated α-Syn, and cytosolic pS129 levels, a model generated by using logistic regression analysis was able to discriminate patients with PD from neurologically normal controls, with a sensitivity and a specificity of 72 and 68%, respectively. CONCLUSIONS: These results suggest that total, aggregated and phosphorylated α-Syn levels are altered in PD erythrocytes and peripheral erythrocytic α-Syn is a potential PD biomarker that needs further validation.

Alpha-Synuclein in Peripheral Tissues in Parkinson's Disease.

Parkinson's disease (PD) is a common neurodegenerative disorder. To date, the diagnosis of PD relies mainly on clinical manifestations whereas neuropathological confirmation of the brain is only possible with postmortem studies. Neuronal loss in the substantia nigra pars compacta (SNc) associated with Lewy bodies/neurites is the pathological hallmark feature of PD. The major component of Lewy pathology (LP) is misfolded alpha-synuclein (α-SYN). There is evidence that the distribution of LP is not only limited to the brain but extends to peripheral tissues, including gastrointestinal tract, salivary glands, olfactory mucosa, skin, retina, adrenal gland, and heart. Sensitivity and specificity of α-SYN detection in PD vary greatly among studies due to methodological heterogeneity, such as sampling sites and size, tissue preparation, staining techniques, and antibodies used. Of note, α-SYN has also been found in preclinical and prodromal PD. Further in vivo studies focusing on favorable biopsy sites and standard techniques are needed to get better understanding of α-SYN deposits in preclinical, prodromal, and clinical PD.

Alpha-synuclein in erythrocyte membrane of patients with multiple system atrophy: A pilot study.

BACKGROUND: Multiple system atrophy(MSA) is a neurodegenerative disease characterized by intracellular α-synuclein deposits. There is an unmet need for blood-based biomarkers to diagnose MSA. Our previous studies have reported elevated α-synuclein levels in erythrocytes of MSA patients. However, α-synuclein protein in the membrane and cytoplasm of erythrocytes in MSA have not been investigated. METHODS: The membrane and cytoplasm were extracted from erythrocytes in 77 patients with MSA and 133 healthy controls. Levels of total and oligomeric α-synuclein were detected using Electrochemiluminescence assays. The correlations between α-synuclein levels and clinical characteristics were explored in MSA group. The diagnostic value of erythrocyte α-synuclein for MSA was determined by Receiver operator characteristic curve. RESULTS: α-synuclein levels in the erythrocyte membrane were significantly elevated in MSA patients compared with the healthy controls (total α-synuclein, p = 0.003; oligomeric α-synuclein/total α-synuclein, p = 0.033; oligomeric α-synuclein/protein, p < 0.001). The combination of total and oligomeric α-synuclein levels in erythrocyte membrane could efficiently distinguish MSA from healthy controls (sensitivity of 79.2%; specificity of 69.2%; area under the curve: 0.771). In contrast, no significant difference was found in erythrocyte cytoplasm α-synuclein levels. In the subgroup of 48 patients with probable MSA, there was a weakly negative correlation between oligomeric α-synuclein/protein in erythrocyte membrane and disease duration (r = -0.336; p = 0.009). CONCLUSION: Our pilot study suggests that the membrane fraction of α-synuclein levels in erythrocyte were elevated in patients with MSA, and these levels may be decreased with the development of disease.

α-Synuclein in salivary extracellular vesicles as a potential biomarker of Parkinson's disease.

BACKGROUND: Detection of α-synuclein (α-syn) in biological fluids such as saliva may serve as potential biomarker of PD. α-syn pertaining to extracellular vesicles (EVs) has been recently studied in plasma, but not in other biological fluids such as saliva. AIM: 1) To investigate the presence of exosomes in PD saliva; 2) to explore the value of α-syn in salivary EVs as potential biomarker in PD. METHODS: Saliva samples were obtained from 74 PD and 60 healthy controls (HCs). The EVs were extracted from saliva by XYCQ EV Enrichment KIT. Western blot and Nanosight 300 were used to validate the existence of exosomes in EVs and to analyze the size of salivary EVs. Salivary EVs α-syn levels, including total α-syn (α-synTotal), oligomeric α-syn (α-synOlig) and phosphorylated-ser129 α-syn (α-synPS129), were measured by Electrochemiluminescence (ECL) assays. Diagnostic value and clinical relevance of salivary EVs α-syn were assessed by Receiver Operator Characteristic (ROC) curve and Spearman correlation. RESULTS: Alix and CD9 positive EVs, representing the presence of exosomes, was detected in PD salivary samples. Size of salivary EVs was about 30-400 nm. The levels of α-synOlig and α-synOlig/α-synTotal in the salivary EVs were higher in PD than in HCs (10.39 ± 1.46 pg/ng vs.1.37 ± 0.24 pg/ng, p＜0.001;1.70 ± 0.52 pg/ng vs.0.67 ± 0.26 pg/ng, p＜0.001). There was no significant difference in α-synTotal、α-synPS129、 α-synPS129/α-synTotal ratio between PD and HCs (P = 0.723, 0.634, 0.734, respectively). α-synOlig 2.05 pg/ng distinguished PD from HCs with sensitivity 92% and specificity 86%; α-synOlig /α-synTotal 0.18 pg/ng differentiated PD from HCs with sensitivity 81% and specificity 71%. No significant correlation between salivary EVs α-synOlig, α-synOlig/α-synTotal and disease severity was found. CONCLUSIONS: Exosomes are present in PD saliva. The α-synOlig and α-synOlig/α-synTotal ratio in salivary EVs may serve as potential diagnostic biomarkers for PD.

Auricular Vagus Nerve Stimulation Exerts Antiinflammatory Effects and Immune Regulatory Function in a 6-OHDA Model of Parkinson's Disease.

According to epidemiologic studies, smoking appears to downregulate the prevalence of Parkinson's disease (PD), possibly due to antiinflammatory mechanisms via activation of α7 nicotinic acetylcholine receptors (α7 nAChRs). This receptor also appears to play a role in T-cell differentiation. Recently, it has become apparent that the innate immune system participates in PD pathogenesis. The aim of this study was to evaluate the effects of auricular vagus nerve stimulation (aVNS) on substantia nigra (SN) dopaminergic neurodegeneration and the associated neuroinflammation and immune responses in a rat PD model. Adult male Wistar rats were unilaterally administered 6-hydroxydopamine (6-OHDA) to the medial forebrain bundle, followed by aVNS treatment after surgery. Following motor behavioral tests, the expression of tyrosine hydroxylase (TH) in the SN and the levels of inflammatory cytokines in the ventral midbrain were evaluated. In addition, changes in the trends of subsets of CD4+ T lymphocytes in the SN were measured by immunofluorescence staining. Western blotting was used to evaluate the α7 nAChR protein level. Compared with 6-OHDA treats rats, aVNS treatment significantly improved motor deficits, increased TH and α7 nAChR expression, and reduced the levels of inflammatory cytokines (tumor necrosis factor-a (TNF-α) and interleukin-1ß (IL-1ß)) (p < 0.05). Additionally, aVNS increased the numbers of regulatory T (Treg) cells while decreasing T helper (Th)17 cells. aVNS exerted neuroprotective effects against dopaminergic damage, possibly by suppressing the evolution of inflammation and modulating innate immune responses. Thus, aVNS may be a potential promising therapy in the future.

Frequency-dependent effects of subthalamic deep brain stimulation on motor symptoms in Parkinson's disease: a meta-analysis of controlled trials.

This study aims to investigate how the frequency settings of deep brain stimulation (DBS) targeting the subthalamic nucleus (STN) influence the motor symptoms of Parkinson's disease (PD). Stimulation with frequencies less than 100 Hz (mostly 60 or 80 Hz) is considered low-frequency stimulation (LFS) and with frequencies greater than 100 Hz (mostly 130 or 150 Hz) is considered high-frequency stimulation (HFS). We conducted a comprehensive literature review and meta-analysis with a random-effect model. Ten studies with 132 patients were included in our analysis. The pooled results showed no significant difference in the total Unified Parkinson Disease Rating Scale part III (UPDRS-III) scores (mean effect, -1.50; p = 0.19) or the rigidity subscore between HFS and LFS. Compared to LFS, HFS induced greater reduction in the tremor subscore within the medication-off condition (mean effect, 1.01; p = 0.002), while no significance was shown within the medication-on condition (mean effect, 0.01; p = 0.92). LFS induced greater reduction in akinesia subscore (mean effect, -1.68, p = 0.003), the time to complete the stand-walk-sit (SWS) test (mean effect, -4.84; p < 0.00001), and the number of freezing of gait (FOG) (mean effect, -1.71; p = 0.03). These results suggest that two types of frequency settings may have different effects, that is, HFS induces better responses for tremor and LFS induces greater response for akinesia, gait, and FOG, respectively, which are worthwhile to be confirmed in future study, and will ultimately inform the clinical practice in the management of PD using STN-DBS.

Microbial Community Analyses of the Deteriorated Storeroom Objects in the Tianjin Museum Using Culture-Independent and Culture-Dependent Approaches.

In the storeroom C7 of the Tianjin Museum, one wooden desk and two leather luggages dated back to Qing dynasty (1644-1912 AD) presented viable microbial contamination. The aim of the present study was to investigate microbial communities responsible for the biodeterioration of storeroom objects using a combination of culture-independent and culture-dependent methods as well microscopic techniques. Scanning electron microscopy (SEM) revealed that the microflora on three storeroom objects were characterized by a marked presence of Eurotium halophilicum. Real-time quantitative polymerase chain reaction (qPCR) analysis proved that fungi were the main causative agents behind the biodeterioration in this case. Fungal internal transcribed spacer (ITS) amplicon sequencing documented the presence of two main fungi - Eurotium halophilicum and Aspergillus penicillioides. Molecular identification of fungal strains isolated from the surfaces and the air of the storeroom were most closely related to Chaetomium, Aspergillus, Penicillium, and Fusarium, showing discrepancies in fungal taxa compared to ITS amplicon sequencing. The most isolated bacterial phylum was Firmicutes, mostly Bacillus members. In addition, four biocide products - Preventol® D 7, P 91, 20 N and Euxyl® K 100 were selected to test their capability against fungal strains isolated from the surfaces. According to the susceptibility assay, Preventol® D 7 based on isothiazolinones was the most effective against fungal isolates. Findings from this study provided a knowledge about storeroom fungi, and exemplify a type of preliminary test that may be conducted before planning any biocide treatment, which may be useful to mitigate the fungal deterioration for further conservation of the museum.