Introduction: Brain-derived neurotrophic factor (BDNF) plays an important role in the survival of dopaminergic neurons. Clinical studies have suggested that serum BDNF levels are reduced in patients with Parkinson's disease (PD). However, no study has investigated peripheral BDNF levels and BDNF Val66Met polymorphism in the prodromal stage of PD and their relationship with disease conversion. Methods: In total, 120 patients with video-polysomnography confirmed isolated REM sleep behavior disorder (iRBD) and 120 healthy controls (HCs) were enrolled. Genetic analyses were performed, and plasma levels of BDNF were measured. All patients with iRBD underwent comprehensive clinical testing, and 107 iRBD patients were prospectively followed up. Results: Plasma BDNF levels were significantly lower in the iRBD group than in HCs (18,878.85 pg/mL vs. 24,649.85 pg/mL, p = 0.002), but no differences were observed in BDNF Val66Met carrier rates between the two groups. Plasma BDNF levels did not differ significantly between BDNF Val66Met carriers and noncarriers. Notably, higher plasma BDNF levels were associated with an increased risk of short-term disease conversion (hazard ratio = 3.418, 95% CI: 1.520-7.684, p = 0.003), whereas BDNF Val66Met carrier rates showed no such association. Conclusion: Our findings suggest that plasma BDNF is significantly associated with iRBD and may likely serve as a prognostic biomarker for the development of neurodegenerative disease. However, the BDNF Val66Met polymorphism may not be involved in the pathogenesis of iRBD as well as phenoconversion in the studied population.

Introduction: Brain-derived neurotrophic factor (BDNF) plays an important role in the survival of dopaminergic neurons. Clinical studies have suggested that serum BDNF levels are reduced in patients with Parkinson’s disease (PD). However, no study has investigated peripheral BDNF levels and BDNF Val66Met polymorphism in the prodromal stage of PD and their relationship with disease conversion. Methods: 120 patients with video-polysomnographically (v-PSG) confirmed iRBD and 120 healthy controls (HCs) were enrolled. Genetic analyses were performed, and plasma levels of BDNF were measured. All patients with iRBD underwent comprehensive clinical testings, and 107 iRBD patients were prospectively followed-up. Results: Plasma BDNF levels were significantly lower in the iRBD group than in HCs (18878.85 pg/ml vs. 24649.85 pg/ml, p = 0.002), but no differences were observed in BDNF Val66Met carrier rates between the two groups. Plasma BDNF levels did not differ significantly between BDNF Val66Met carriers and non-carriers. Notably, higher plasma BDNF levels were associated with an increased risk of short-term disease con-version (Hazard Ratio = 3.418, 95% CI 1.520-7.684, p = 0.003), whereas BDNF Val66Met carrier rates showed no such association. Conclusion: Our findings suggest that plasma BDNF is significantly associated with iRBD and may likely serve as a prognostic biomarker for the development of neurodegenerative dis-ease. However, the BDNF Val66Met polymorphism may not be involved in the path-ogenesis of iRBD as well as phenoconversion in the studied population.

Abstract Small extracellular vesicles (EVs) are nanosized vesicles (< 200 nm) secreted from various tissues, including the central nervous system (CNS), into diverse biofluids. Due to their ability to carry molecular cargo that reflects the physiological state of their parental cells, small EVs represent promising diagnostic carriers for neurodegenerative diseases, such as Alzheimer’s disease (AD) and Parkinson’s disease (PD). While biofluid-derived small EVs offer a “liquid biopsy” solution, their clinical translation is severely hindered by the limitations of conventional isolation methods, which are often time-consuming, costly, or yield low purity. Building upon the “ExoPRISM” (Exosome Precipitation by Ionic Strength Modulation) framework, we developed and validated an optimized, cost-effective ammonium sulfate (AS)-based pipeline for small EVs isolation. We have refined and established the optimal AS ratio specifically for plasma and further demonstrated its feasibility for isolating small EVs from saliva. Subsequently, we introduced a targeted purification step using glutamate aspartate transporter (GLAST) antibodies to specifically isolate central nervous system (CNS)-derived EVs. This step robustly demonstrated that our AS-based approach preserves small EVs integrity and enables the effective isolation of Astrocyte-Derived Extracellular Vesicles (ADEVs) for downstream applications. Finally, the practical utility of this optimized protocol was validated in a clinical cohort. Our findings highlight the robustness, high efficiency, and significant translational potential of this AS-based method for the early and accurate diagnosis of neurodegenerative diseases. We optimized the AS concentration for small EVs precipitation, ultimately identifying 2.66 M as the optimal working concentration. This method demonstrated remarkable time efficiency, completing the entire isolation process in approximately 90 min. Furthermore, it only requires centrifugation at 12,000 xg, significantly enhancing its practicality in clinical diagnostic scenarios. To expand its potential application scope, we also tested the method’s efficacy on other bodily fluids and found that the concentration of 2.66 M was equally effective in isolating small EVs from saliva. Comparative analyzes against the commercially available ExoQuick kit demonstrated that our AS-based precipitation method achieved comparable efficacy in isolating small EVs from the plasma of AD patients and in purifying ADEVs. Furthermore, comprehensive validation using nanoparticle tracking analysis (NTA), electron microscopy, and detection of canonical small EVs markers (CD63, CD9) confirmed the functional equivalence of the two isolation methods. The cumulative evidence from this study firmly establishes the AS-based small EVs isolation protocol as a viable and robust efficacy comparable to commercially available kits. This method confers three distinct advantages: enhanced temporal efficiency, reduced cost burden, and minimal interference with downstream assays, rendering it highly amenable to clinical implementation. Notably, it enables the effective isolation of small EVs from plasma, a conventional biofluid widely utilized in clinical diagnostics, as well as from saliva. These attributes underscore the method’s potential in advancing the early detection of neurodegenerative disorders. Looking ahead, the versatility of this approach suggests its applicability across a diverse array of biological specimens, thereby facilitating the expansion of small EVs-based diagnostic research and clinical practice.

The human circadian rhythm system is governed by a transcriptional-translational feedback loop of clock genes, including Bmal1, which exhibits a near-24-hour oscillatory period. Emerging evidence highlights the critical contribution of circadian genes to stroke pathogenesis and outcomes, yet mechanistic insights into their regulatory roles remain limited. To bridge this knowledge gap, we aimed to determine whether the serum of stroke patients could affect changes in the molecular clock and whether such changes in the molecular clock were potentially linked to abnormal brain functional connectivity in stroke patients. This prospective case-control study included 17 patients with stroke and 17 age-matched healthy participants. All participants were required to complete a standardized comprehensive questionnaire. Transfected human osteosarcoma cells (U2OS) stably expressing Bmal1-dluc reporters were treated with serum from both groups to monitor changes in the daily oscillation period of Bmal1. We also compared the changes in resting-state hypothalamic intrinsic functional connectivity(FC) between the two groups. Further, we explored the correlation between the differences in FC between the two groups and the daily oscillation period of Bmal1. Compared to the healthy control group, stroke patients exhibited a significantly prolonged peripheral Bmal1 circadian oscillation period (24.32 ± 0.41 h vs. 23.81 ± 0.48 h, t = 3.352, p = 0.002). In FC analysis, the stroke group showed enhanced connectivity from the hypothalamus to the right occipital lobe and the right insular region. Notably, the degree of this connectivity enhancement demonstrated a significant positive correlation with the length of Bmal1 oscillation period (r = 0.369, p = 0.032; r = 0.393, p = 0.022). Our findings suggest that serum from stroke patients lengthens the circadian period in U2OS cells and may be associated with alterations in brain functional connectivity. However, the small sample size limits statistical power, and these exploratory results require replication in larger independent cohorts. ChiCTR2000031671(April 4, 2020).

Neutrophil-mediated neuroinflammation plays a crucial role in secondary brain injury following severe cerebral venous thrombosis (CVT). Although previous studies have reported that the combination of glucocorticoids (GCs) and anticoagulation is associated with improved clinical outcomes, its mechanism remains unknown. We hypothesized that the combination therapy may exert benefit by modulating neutrophil-driven inflammation. This study included a cohort of 50 patients diagnosed with severe CVT who were undergoing treatment with the combination therapy. We investigated the dynamic alterations in the NLRP3/NETosis inflammatory process by analyzing paired serum and cerebrospinal fluid (CSF) samples collected at baseline and 1 week post-treatment. Neurological function was systematically evaluated using the National Institutes of Health Stroke Scale (NIHSS) and the modified Rankin Scale (mRS). The combined therapy was associated with reduced CSF levels of key NLRP3/NETosis mediators, including NOD-like receptor family pyrin domain containing 3 (NLRP3), polymorphonuclear neutrophil elastase (PMN Elastase), myeloperoxidase (MPO), and citrullinated histone H3 (CitH3), while the corresponding serum levels were unchanged. Baseline CSF levels of NLRP3, PMN Elastase, and MPO strongly correlated with admission NIHSS and mRS. Early reductions in these central markers were associated with neurological improvement at discharge (ΔNIHSS). Moreover, patients with unfavorable outcomes (discharge mRS > 1) had significantly higher baseline NIHSS and CSF NLRP3 levels. The combined therapy may alleviate severe CVT by modulating the central NLRP3/NETosis inflammatory process.

BACKGROUND:Previous studies have suggested that metabolic alterations are involved in the pathogenesis of α-synucleinopathies. However, the metabolic changes in isolated REM sleep behavior disorder (iRBD), the prodromal stage of α-synucleinopathies, and their relationship to phenoconversion remain unexplored. OBJECTIVE:To investigate the serum metabolic markers in patients with iRBD and evaluate their association with the risk of conversion to α-synucleinopathies. METHODS:We enrolled 148 patients with video-polysomnography (vPSG)-confirmed iRBD and 142 age- and sex-matched healthy controls (HCs). 138 patients with iRBD were prospectively followed over an average duration of 3.67 years and 34 developed to α-synucleinopathies. Serum metabolic markers, including uric acid (UA), fasting blood glucose (FBG), homocysteine (Hcy), and lipid profile were measured at baseline. RESULTS:Compared with HCs, patients with iRBD exhibited significantly lower serum UA (332.15 μmol/L vs. 314.73 μmol/L, p = 0.010) and FBG (6.02 mmol/L vs. 5.69 mmol/L, p = 0.008) levels. Competing-risk regression analysis revealed that lower serum UA (hazard ratio [HR] 0.992, 95% CI 0.985-0.998, p = 0.013), higher Hcy (HR 1.070, 95% CI 1.028-1.113, p = 0.001), and higher FBG (HR 1.477, 95% CI 1.081-2.017, p = 0.014) were associated with an increased risk of conversion to dementia. CONCLUSION:Lower serum UA and FBG levels are potential risk factors for iRBD. Furthermore, distinct markers, including lower serum UA levels and elevated Hcy and FBG levels, were associated with an increased risk of dementia conversion. Our findings suggest that metabolic markers may be valuable indicators for predicting specific phenoconversion outcomes in iRBD.

Cell-free DNA (cfDNA) biomarkers derived from Arthrobacter luteus (ALU) repeats and long interspersed nuclear elements 1 (LINE1) - including ALU-115, ALU-247, LINE1-97, and LINE1-266 concentrations, as well as the integrity ratios ALU-247/115 and LINE1-266/97 - are commonly utilized to assess cfDNA quantity and integrity. This study examined the impact of delayed blood processing and prolonged plasma storage on these biomarkers using quantitative polymerase chain reaction. Blood samples were collected from twelve healthy individuals (6 males; mean age, 65.8 +/- 4.69 years) into dipotassium ethylenediaminetetraacetic acid tubes. Plasma cfDNA was extracted after various storage durations and temperatures, with aliquots from immediately processed blood subsequently stored at -80 degrees C for different time intervals. Except for LINE1-97, most biomarkers showed significantly higher levels in plasma isolated from whole blood stored at room temperature compared to plasma processed immediately. Storage at 4 degrees C resulted in fragment-specific effects: ALU-247/115 levels remained stable at 3 hours but decreased at 6 hours, while LINE1-266/97 levels increased at both time points. For plasma stored at -80 degrees C, ALU-derived biomarkers remained stable for up to 12 months; however, LINE1-97 levels significantly declined, accompanied by a corresponding increase in LINE1-266/97 as early as one month after freezing. These findings indicate that both storage duration and temperature significantly impact the measured levels of ALU- and LINE1-derived cfDNA biomarkers. Consequently, standardization of pre-analytical handling of blood and plasma is crucial for studies evaluating cfDNA quantity and integrity.

Clinical diagnosis of dementia, with Alzheimer's disease (AD) as the major form, relies heavily on memory tests and the caregiver descriptions, both of which are subjective. The discovery of reliable biomarkers may facilitate objective diagnosis. The protein clusterin (CLU), encoded by a well-established AD risk gene, is consistently elevated in AD patients, but its biomarker utility is limited by high interindividual variability. As CLU is produced in most organs and tissues, quantifying CLU secreted specifically from the brain into the bloodstream may help the development of new diagnostic methods. CLU in blood can be phosphorylated at T393-S394 and/or S396, but these phosphorylations are absent in brain parenchyma. Peripherally administered non-phosphorylated CLU has been shown to reduce neuroinflammation and AD pathology in mouse models. A monoclonal antibody, 3D3F10, targeting non-phosphorylated CLU at T393-S394 or S396 was generated. This antibody showed an ability to distinguish serum samples of dementia and non-dementia (p < 0.001, AUC = 0.897, sensitivity: 81.8%, specificity: 95.5%, Youden index: 0.77), but did not distinguish Parkinson's disease. The direction of the change contradicted our initial hypothesis, and further analyses suggested that phospho-CLU in dementia patients is unlikely to originate from the brain. These results established 3D3F10 as a novel tool for modification-specific CLU detection, indicated a potential of non-phospho-CLU as a biomarker for dementia, and peripheral phospho-CLU might play a role in pathogenesis.

Idiopathic rapid eye movement sleep behavior disorder (iRBD) is a well-established prodromal manifestation of α-synucleinopathies, with two principal phenoconversion trajectories: parkinsonism-first and dementia-first. Although subtle gait abnormalities have been observed prior to phenoconversion, their predictive value remains unclear. We investigated whether wearable sensor-based gait parameters are associated with phenoconversion and its clinical trajectories in iRBD. Sixty-eight polysomnography-confirmed iRBD patients and 61 healthy controls were enrolled at baseline. The iRBD cohort was followed for a mean of 3.68 years, with 21 patients converting to neurodegenerative diseases; 38 patients completed follow-up gait assessment. Participants performed one-minute walking trials under normal, fast, and dual-task conditions while wearing six inertial sensors. At baseline, iRBD patients exhibited significant gait abnormalities compared with controls. Several gait parameters were more strongly associated with subsequent conversion to Parkinson's disease (PD) than to dementia with Lewy bodies (DLB), including shorter stride length, greater swing time variability, reduced arm swing range, increased arm swing variability, and lower peak arm swing velocity. Longitudinally, converters showed a steeper decline in stride length and greater increases in dual-task cost. Wearable sensor-based gait assessment may serve as a digital biomarker for predicting phenoconversion in iRBD, particularly along the parkinsonism-first trajectory.

BACKGROUND:The accumulation of α-synuclein (SNCA) in the central nervous system is a hallmark of Parkinson's disease (PD) and multiple system atrophy (MSA). SNCA intron 1 methylation is implicated in SNCA transcriptional regulation and may serve as a peripheral epigenetic signal in synucleinopathies. However, studies of SNCA methylation in leukocyte-derived DNA have yielded inconsistent results. We aimed to evaluate whether cell-free DNA (cfDNA)-based SNCA intron 1 methylation differs in PD or MSA compared with normal controls (NC). METHODS:Plasma cfDNA was collected from 105 patients with PD, 50 with MSA, and 114 NC. DNA methylation at CpG sites 10-17 was quantified by bisulfite pyrosequencing. Multivariable linear and logistic regression models, adjusted for age, sex, and education, were used to compare methylation levels and estimate odds ratios (ORs). RESULTS:Patients with PD exhibited hypermethylation at CpG site 14 and higher mean methylation across CpG sites 10-17 compared with NC. Patients with MSA showed hypermethylation at CpG sites 10, 12, 13, and 17 and elevated mean methylation. Elevated mean methylation was also observed in drug-naïve de novo PD and early-stage PD patients. Compared with the lowest tertile, the highest mean methylation tertile was associated with increased odds of PD (OR, 2.49; 95% CI, 1.08-5.92) and MSA (OR, 5.02; 95% CI, 1.58-18.00). CONCLUSION:Plasma cfDNA SNCA intron 1 hypermethylation is associated with PD and MSA and detectable in drug-naïve and early-stage PD. It may represent a peripheral epigenetic alteration and warrants evaluation as an adjunctive signal for early screening.

Mitochondrial dysfunction is central to Parkinson’s disease (PD), but assessing it in vivo remains challenging. Plasma L1CAM-immunocaptured putative neuron-derived exosomes (NDEs) offer minimally invasive access to brain molecular signatures. This study investigated whether mitochondrial complex (MC) proteins in NDEs are altered in PD and explored their association with clinical features. Plasma putative NDEs were isolated from 28 patients with PD and 33 normal controls (NCs) by L1CAM immunocapture. Levels of mitochondrial subunits—NDUFS3 (Complex I), UQCRC2 (Complex III), MT-CO1 (Complex IV), and ATP5F1A (Complex V)—and the antioxidant enzyme SOD1 were quantified by ELISA. Correlations with clinical severity and diagnostic performance were analyzed. Compared with NCs, PD patients exhibited significantly lower levels of NDUFS3 and UQCRC2 in NDEs (p < 0.05, after FDR correction). NDUFS3, UQCRC2, and SOD1 showed modest inverse correlations with motor symptom severity (R = −0.26). The NDUFS3/UQCRC2 combination yielded an AUC of 0.763 (95

This study explores the effect of risk factors on the progression of idiopathic rapid eye movement (REM) sleep behavior disorder (iRBD) to α-synucleinopathies in a Chinese cohort. Patients with iRBD were enrolled and assessed for environmental factors and lifestyle using standardized structured questionnaires at baseline. All patients were prospectively followed for phenoconversion monitoring. The cumulative incidence was estimated using survival analysis. Of 155 iRBD enrolled in the cohort, follow-up information was available in 141 patients. The phenoconversion rate was 16.3% after 3 years, 27.6% after 5 years, and 57.2% after 10 years. Eighteen participants converted within 3 years, 27 converted within 5 years, and 36 converted within 10 years. IRBD with positive family history of parkinsonism had an increased risk of being converted to α-synucleinopathies, while tea drinking was associated with a decreased phenoconversion risk. Our findings shed light on a potential application of tea drinking in modifying iRBD progression.

Circadian disruptions are increasingly recognized in Alzheimer's disease (AD) patients and may influence disease onset and progression. This study examines how AD pathology affects blood-borne factors that regulate circadian rhythms. Eighty-five participants from the Sino Longitudinal Study on Cognitive Decline were enrolled: 35 amyloid-beta negative normal controls (Aβ− NCs), 23 amyloid-beta positive normal controls (Aβ+ NCs), 15 patients with amnestic mild cognitive impairment (aMCI), and 12 with Alzheimer's disease dementia (ADD). Patients with aMCI and ADD were grouped as cognitively impaired (CI). Cellular circadian period length was assessed using a serum-based assay. Expression levels of clock genes in serum-treated cells and in leukocytes of participants were measured via real-time PCR. Plasma biomarkers were quantified using a single-molecule array immunoassay. Pineal parenchymal and hippocampal volumes were determined by magnetic resonance imaging. The cellular circadian period length was significantly extended by serum from CI patients than by that from Aβ− NCs (p < 0.01). Treatment of cells with serum from the CI patients resulted in suppressed expression of the clock genes Bmal1 and Nr1d1. Strong relationships between the expression levels of clock genes observed in leukocytes of the Aβ− NC group did not appear in those of the Aβ+ NC or CI groups. The significant correlation of cellular circadian period length and the pineal volume was only observed in the Aβ− NC group, but not in the Aβ+ NC or CI groups. This study indicates the presence of significant changes in blood-borne factors that could affect the circadian rhythms in AD, starting even at preclinical stages. These alterations could precede cognitive decline and contribute to AD pathogenesis. The cohort is registered at ClinicalTrials.gov (SILCODE: NCT03370744; Registered on Mar 15th, 2017).

As an inflammatory disease, atherosclerosis is associated with acute ischemic stroke (AIS), but its early identification and intervention efficacy remain suboptimal. A new research direction may be to explore peripheral atherosclerotic biomarkers from the perspective of mitochondrial dysfunction, which can induce inflammatory cell activation. Moreover, the degree of overall cervicocephalic atherosclerosis (namely, atherosclerotic burden) is more closely related to AIS prognosis than local atherosclerotic lesions. Therefore, this study investigated the relationship between mitochondrial dysfunction in peripheral blood mononuclear cells (PBMCs), including monocytes and lymphocytes, and overall cervicocephalic atherosclerotic burden and AIS outcome. Patients with AIS and cervicocephalic atherosclerosis were enrolled and followed up for 90 days. The reactive oxygen species (ROS) and the mitochondrial deoxyribonucleic acid copy number (mtDNA-CN) in PBMCs were measured respectively through a fluorescence probe and a droplet digital polymerase chain reaction to evaluate mitochondrial function. The overall intracranial and cervical atherosclerotic burden (ICAB) was quantified by summing up the atherosclerosis degree points in each arterial segment as assessed by computed tomography angiography. A modified Rankin Scale (mRS) score >2 was considered a 90-day unfavorable functional outcome. Five (4.9%) of the 103 patients with AIS were lost to follow-up. mtDNA-CN [adjusted β = -0.099, 95% confidence intervals (CIs) = -0.153 ∼ -0.044, p < 0.001] and ROS content (adjusted β = 1.275, 95%CI = 0.885 ∼ 1.665, p < 0.001) were correlated with ICAB. The risk of a 90-day unfavorable functional outcome increased with higher ROS content [adjusted odds ratio (OR) = 1.523, 95%CI = 1.172 ∼ 1.981, p = 0.002] and decreased with higher mtDNA-CN (adjusted OR = 0.911, 95%CI = 0.850 ∼ 0.976, p = 0.008). PBMC mitochondrial dysfunction was found to be independently associated with extensive and severe cervicocephalic atherosclerosis and a 90-day unfavorable functional outcome in patients with AIS, which may provide a novel approach to improving the early identification and risk stratification of cervicocephalic atherosclerosis, along with the prediction of the outcome of atherosclerotic AIS.

Introduction : Telomere length and mitochondrial DNA (mtDNA) biomarkers (mtDNA copy number and mtDNA common deletion) are considered to be involved in age-related disorders and neurodegenerative diseases. The relationship and potential mechanism between these three aging-biomarkers and Parkinson’s disease (PD) is not yet fully understood. Method : This study aimed to measure the peripheral blood leukocyte telomere length, mtDNA copy number and mtDNA common deletion by quantitative polymerase chain reaction in PD patients and healthy controls. A total number of 64 PD patients were recruited from the Department of Neurology, Beijing Xuanwu Hospital for this study, and 65 controls were recruited from the community cohort. All individuals are nonsmokers and underwent neuropsychological tests to assess cognitive function. All patients underwent Hoehn-Yahr stages and Movement Disorder Society Unified Parkinson’s Disease Rating Scale Part 2/3. Results : The result showed that there were no significant differences about these three aging biomarkers between the PD patients and controls. However, the spearman correlation analysis showed that the telomere length was negatively correlated with age (PD: p=0.007; Control: p=0.021) and positively correlated with mtDNA copy number both in PDs (p<0.001) and healthy controls (p=0.016). In PD group, mtDNA common deletion was positively correlated with age (p=0.001) and was also positively correlated with mtDNA copy number (p=0.023). Discussion : There is a potential interaction between the telomere length and mtDNA copy number in all population. However, telomere length and mtDNA biomarkers might not be used as the risk factor for PD. With the increase of age in PD, the cell senescence and the mitochondrial dysfunction are aggravated, and the mtDNA common deletion is overloaded. Meanwhile, the mtDNA copy number is also increased with the increased mtDNA commom deletion, which may be a compensatory mechanism to maintain the stability of mitochondrial function under PD pathological conditions.

Background: Cognitive impairment is common in patients with Parkinson’s disease (PD) and occurs through multiple mechanisms, including Alzheimer’s disease (AD) pathology and the involvement of α-synucleinopathies. We aimed to investigate the pathological biomarkers of both PD and AD in plasma and neuronal extracellular vesicles (EVs) and their association with different types of cognitive impairment in PD patients. Methods: A total of 122 patients with PD and 30 healthy controls were included in this cross-sectional cohort study between March 2021 and July 2023. Non-dementia PD patients were divided into amnestic and non-amnestic groups according to the memory domain of a neuropsychological assessment. Plasma and neuronal EV biomarkers, including α-synuclein (α-syn), beta-amyloid (Aβ), total tau (T-tau), phosphorylated tau181 (p-tau181), and glial fibrillary acidic protein (GFAP), were measured using a single-molecule array and a chemiluminescence immunoassay, respectively. Results: Neuronal EV but not plasma α-syn levels, were significantly increased in PD as compared to healthy controls, and they were positively associated with UPDRS part III scores and the severity of cognitive impairment. A lower plasma Aβ42 level and higher neuronal EV T-tau level were found in the amnestic PD group compared to the non-amnestic PD group. Conclusions: The results of the current study demonstrate that neuronal EV α-syn levels can be a sensitive biomarker for assisting in the diagnosis and disease severity prediction of PD. Both AD and PD pathologies are important factors in cognitive impairment associated with PD, and AD pathologies are more involved in amnestic memory deficit in PD.

In clinical specialties focusing on neurological disorders, there is a need for comprehensive and integrated non-invasive, sensitive, and specific testing methods. Both Parkinson’s disease and multiple system atrophy are classified as α-synucleinopathies, characterized by abnormal accumulation of α-synuclein protein, which provides a shared pathological background for their comparative study. In addition, both Parkinson’s disease and multiple system atrophy involve neuronal death, a process that may release circulating cell–free DNA (cfDNA) into the bloodstream, leading to specific alterations. This premise formed the basis for investigating cell–free DNA as a potential biomarker. Cell-free DNA has garnered attention for its potential pathological significance, yet its characteristics in the context of Parkinson’s disease and multiple system atrophy are not fully understood. This study investigated the total concentration, nonapoptotic level, integrity, and cell-free DNA relative telomere length of cell-free DNA in the peripheral blood of 171 participants, comprising 76 normal controls, 62 patients with Parkinson’s disease, and 33 patients with multiple system atrophy. In our cohort, 75.8% of patients with Parkinson’s disease (stage 1–2 of Hoehn & Yahr) and 60.6% of patients with multiple system atrophy (disease duration less than 3 years) were in the early stages. The diagnostic potential of the cell-free DNA parameters was evaluated using receiver operating characteristic (ROC) analysis, and their association with disease prevalence was examined through logistic regression models, adjusting for confounders such as age, sex, body mass index, and education level. The results showed that cell-free DNA integrity was significantly elevated in both Parkinson’s disease and multiple system atrophy patients compared with normal controls (P < 0.001 for both groups), whereas cell-free DNA relative telomere length was markedly shorter (P = 0.003 for Parkinson’s disease and P = 0.010 for multiple system atrophy). Receiver operating characteristic analysis indicated that both cell-free DNA integrity and cell-free DNA relative telomere length possessed good diagnostic accuracy for differentiating Parkinson’s disease and multiple system atrophy from normal controls. Specifically, higher cell-free DNA integrity was associated with increased risk of Parkinson’s disease (odds ratio [OR]: 5.72; 95% confidence interval [CI]: 1.54–24.19) and multiple system atrophy (OR: 10.10; 95% CI: 1.55–122.98). Conversely, longer cell-free DNA relative telomere length was linked to reduced risk of Parkinson’s disease (OR: 0.16; 95% CI: 0.04–0.54) and multiple system atrophy (OR: 0.10; 95% CI: 0.01–0.57). These findings suggest that cell-free DNA integrity and cell-free DNA relative telomere length may serve as promising biomarkers for the early diagnosis of Parkinson’s disease and multiple system atrophy, potentially reflecting specific underlying pathophysiological processes of these neurodegenerative disorders.

Background: Gait disturbance is a vital characteristic of motor manifestation in α– synucleinopathies, especially Parkinson’s disease. Subtle gait alterations are present in isolated rapid eye movement sleep behavior disorder (iRBD) patients before phenoconversion; it is yet unclear, if gait analysis may predict phenoconversion. Objective: To investigate subtle gait alterations and explore whether gait analysis using wearable sensors is associated with phenoconversion of iRBD to α-synucleinopathies. Methods: Thirty-one polysomnography-confirmed iRBD patients and 33 healthy controls (HCs) were enrolled at baseline. All participants walked for a minute while wearing 6 inertial sensors on bilateral wrists, ankles, and the trunk (sternal and lumbar region). Three conditions were tested: (i) normal walking, (ii) fast walking, and (iii) dual-task walking. Results: Decreased arm range of motion and increased gait variation (stride length, stride time and stride velocity) discriminate converters from HCs at baseline. After an average of 5.40 years of follow-up, 10 patients converted to neurodegenerative diseases (converters). Cox regression analysis showed higher value of stride length asymmetry under normal walking condition to be associated with an early conversion of iRBD to α– synucleinopathies (adjusted HR 4.468, 95% CI 1.088– 18.349, p = 0.038). Conclusions: Stride length asymmetry is associated with progression to α– synucleinopathies in patients with iRBD. Gait analysis with wearable sensors may be useful for screening, monitoring, and risk stratification for disease-modifying therapy trials in patients with iRBD.

To the Editor: Accumulating evidence has shown that the preclinical stage of Alzheimer's disease (AD) (i.e., asymptomatic amyloidosis) lasts for decades before the onset of cognitive symptoms, providing a large window for early intervention. Amyloid pathology, the earliest pathological change associated with AD, can be detected in vivo with cerebrospinal fluid (CSF) analysis or positron emission tomography (PET), and its presence is necessary for the diagnosis of preclinical AD (pre-AD). However, both PET scans and CSF analyses are expensive, hampering their use in large-scale screening. Thus, blood-based biomarkers are desirable alternatives, as they are cost-effective and not invasive. Plasma β-amyloid (Aβ) biomarkers, and particularly the Aβ42/Aβ40 ratio, are logical blood-based candidates, as recent studies have shown decreased Aβ42/Aβ40 ratios along the AD continuum.[1] Several studies have also demonstrated the predictive value of plasma Aβ in combination with other variables. However, magnetic resonance imaging (MRI)-based measures of gray matter atrophy are a neurodegenerative AD biomarker, as cortical atrophy can predict subsequent cognitive decline and AD conversion.[2] Recent data have also demonstrated an association between central amyloid and the memory and language domains of the subjective cognitive decline interview (SCD-I).[3] Thus, in this study, we aimed to (1) investigate the plasma Aβ alterations in pre-AD and validate the diagnostic efficiency of plasma Aβ for detecting brain amyloidosis in peripheral pre-AD; and (2) explore the additive effects of subjective cognitive decline (SCD) features and gray matter volume in the plasma Aβ model. A total of 88 right-handed Han Chinese subjects (57 normal control [NC] and 31 pre-AD) were selected from 617 subjects who were enrolled in the Sino Longitudinal Study on Cognitive Decline (SILCODE) study between March 2017 and September 2018 [Supplementary Figure 1, https://links.lww.com/CM9/B722]. All participants had complete clinical examinations, underwent neuropsychological screening and blood sample collections, and had PET and sMRI scans. The inclusion criteria for pre-AD were: (1) asymptomatic amyloidosis; (2) no typical/atypical AD symptoms; (3) normal neuropsychological test performance; and (4) not meeting mild cognitive impairment (MCI)/dementia criteria. NC included those with negative PET scans and normal neuropsychological test results. The exclusion criteria were: (1) severe depression/anxiety; (2) other cognitive decline-causing conditions; (3) history of psychosis or mental growth slowing; and (4) cognitive decline from brain injury. Subjective cognitive functions were evaluated using the SCD-I developed by the Multicenter German Center for Neurodegenerative Diseases (DZNE)-Longitudinal Cognitive Impairment and Dementia Study (DELCODE). It included information about all five SCD-plus features (decline in memory, onset time, concerns, comparison with others, and informant confirmation) in five domains (memory, language, attention, planning, and others). In the present study, we focused on the involved domains. Objective cognitive functions were evaluated using a battery of neuropsychological tests. The Auditory Verbal Learning Test (AVLT)-HuaShan version was used for memory assessment; the semantic Verbal Fluency Test and the Boston Naming Test were used to assess language functions; and the Shape Trails Tests-A and B were used to assess executive function. The Hamilton Depression Rating Scale (HAMD) and the Hamilton Anxiety Rating Scale (HAMA) were also administered to all participants. Blood sample collection, storage, and analysis of the plasma Aβ42/Aβ40 ratio and amyloid-PET (AV45-PET) and MRI acquisition parameters, processing methods, calculation and analysis of standard uptake value rate (SUVR) values are shown in Supplementary Materials, https://links.lww.com/CM9/B722. Demographics, neuropsychological characteristics, and SCD-I domains were compared using t-tests and chi-squared tests. General linear models (GLMs) were controlled for age, sex, education, and apolipoprotein E (ApoE) ε4 status to compare plasma Aβ and brain volumes. Parietal correlation analyses were adjusted for age, sex, and education assessed associations between plasma Aβ and amyloid deposition and gray matter volume. Logistic regression models were used to compare four classification models, and receiver operating characteristic (ROC) curves were used for the evaluation of performance. The Youden Index determined the plasma Aβ42/Aβ40 cutoff value. P <0.05 was considered to be statistically significant. The demographic and clinical characteristics of all 88 participants, as well as between-group differences, were summarized in Supplementary Table 1, https://links.lww.com/CM9/B722. The NC and pre-AD groups had no differences in age, sex, education, ApoE ε4 positivity, or neuropsychological tests. The prevalence and group differences in SCD domains were shown in Supplementary Table 2, https://links.lww.com/CM9/B722. Both groups reported complaints in memory, executive, and language domains, with pre-AD reporting more memory decline (90.3% [28/31] vs. 66.7% [38/57], P = 0.014) and less language decline (9.7% [3/31] vs. 35.1% [20/57], P = 0.010). No group differences were found in planning, attention, or other domains. ROC analysis only used in memory and language domains. Figure 1A showed the group differences in plasma Aβ levels and their associations with brain amyloid deposition. Compared to the NC group, the pre-AD group had significantly lower Aβ42 (GLM F = 14.827, P = 0.002; 12.51 ± 3.89 pg/mL in NC vs. 10.38 ± 2.46 pg/mL in pre-AD) and Aβ42/Aβ40 (GLM F = 9.104, P <0.001; 0.017 ± 0.0006 in NC vs. 0.014 ± 0.0005 in pre-AD) levels. No significant difference was found in plasma Aβ40 levels between two groups (t = 0.228, P = 0.710). Additionally, lower plasma Aβ42/Aβ40 ratios were associated with higher global SUVR (ρ = –0.228, P = 0.037) [Figure 1B]. Plasma Aβ42 was negatively associated with global SUVR at the trend level (ρ = –0.215, P = 0.050) [Figure 1B], but no correlation was found between plasma Aβ40 and SUVR (ρ = –0.008, P = 0.954).Figure 1: (A)Plasma Aβ42 level and plasma Aβ42/Aβ40 ratios in different groups. Plasma Aβ42 and Aβ40 levels were quantified by Mesoscale Diagnostics in 57 NC and 31 pre-AD patients. Analyses were adjusted for age, gender, education, and ApoE ε4 status.(B) Correlations between SUVR and Aβ42/Aβ40 ratios, plasma Aβ42 levels. (C) Global gray matter volume in different gruops. Analyses were adjusted for age, gender, and education.(D) Correlations between global gray matter volume and SUVR. (E) The ROC curves of different features diagnostic model. AD: Alzheimer's disease; ApoE: Apolipoprotein E; AUC: Area under the curve; NC: Normal control; pre-AD: Preclinical AD; ROC: Receiver operating characteristic; SCD: Subjective cognitive decline; SUVR: Standard uptake value rate.Gray matter volumes of the whole brain and the bilateral hippocampi were compared among the two groups after controlling for age, sex, and education. The pre-AD group showed significantly decreased global gray matter volumes compared to the NC group (t = –2.648, P = 0.010) [Figure 1C], but there were no differences in bilateral hippocampal volumes (left hippocampi: t = 1.361, P = 0.177, right hippocampi: t = 0.705, P = 0.483). Partial correlation analysis, which controlled for age, sex, and education, showed a significant, negative association between global gray matter volume and SUVR (ρ = –0.225, P = 0.039) [Figure 1D], while no association was found between hippocampal volumes and SUVR (ρ = 0.343, P >0.1). We trained logistic regression models to classify pre-AD and NC patients using Aβ characteristics as well as global gray matter volume in combination with the two selected SCD domains. Classification performance was evaluated using ROC curves [Figure 1E]. Global gray matter volume could discriminate between pre-AD and NC patients (area under the curve [AUC] = 0.642, 95% confidence interval [CI] = 0.520–0.765), as could plasma Aβ42/Aβ40 ratios (AUC = 0.762, 95% CI = 0.656–0.868). Youden's cut-offs for plasma Aβ42/Aβ40 ratios and gray matter volume were 0.0145 with a sensitivity of 71% and specificity of 79%, and 389.66 cm3 with a sensitivity of 61% and specificity of 67%. Using the cut-off value of 0.0145 for the plasma Aβ42/Aβ40 ratio, the number of patients receiving AV45-PET would be reduced by 57%. Combining the Aβ42/Aβ40 ratio with the SCD domains also significantly improved the classification performance (AUC = 0.835, 95% CI = 0.748–0.922), which was further slightly improved when combined with global gray matter volume (AUC = 0.839, 95% CI = 0.754–0.925). Our study suggested using memory and language SCD domains increased plasma Aβ42/Aβ40 ratio model accuracy in distinguishing pre-AD from NC. SCD-I, especially in memory and language domains, is linked to lower CSF Aβ42 levels and higher amyloid deposition, indicating its potential as an early AD indicator. SCD is associated with tauopathy independent of global Aβ burden, adding to the predictive power of plasma Aβ42/Aβ40 ratio. Our data validate plasma Aβ42/Aβ40 ratio as a pre-AD screening biomarker in Chinese patients (AUC = 0.762). Our findings are comparable with previous results from the FACEHBI study (AUC = 0.681)[4] and the SCIENCe project (AUC = 0.680).[5] Atrophy has been defined as a biomarker of neurodegeneration following amyloid pathology in AD. In line with previous studies, we found decreased global gray matter volume in pre-AD patients compared to controls. However, there were no significant differences found in bilateral hippocampal volumes between the two groups. Our previous study in individuals at risk for AD also showed reduced global surface areas and cortical volumes but no differences in network or regional scales in patients at risk for AD compared to NC. These findings indicate that changes in gray matter volume in the preclinical stages may be slight but extensive. Limitations include a small cross-sectional sample and exclusion of other diseases that may cause cognitive decline. Plasma Aβ levels may be linked to other comorbidities, and their ability to distinguish AD from other dementias requires further investigation. Pathologic tau biomarkers such as CSF levels of phosphorylated tau or tau PET were not examined. In conclusion, our results suggest that the plasma Aβ42/Aβ40 ratio could reflect central amyloid AD pathology in cognitively normal elderly Chinese subjects, especially when combined with SCD characteristics and neurodegenerative biomarkers. Funding This work was supported by grants from the National Natural Science Foundation of China (Nos. 82020108013 and 82001773), National Key Research and Development Program of China (No. 2022YFC24069004), Beijing Brain Initiative from Beijing Municipal Science & Technology Commission (Z201100005520018), and STI2030-Major Projects (No. 2022ZD0211800). Conflicts of interest None.

Neurofilament light chain (NFL) in blood has been identified as a valuable biomarker in multiple system atrophy (MSA), but data regarding its utility in the early diagnosis and prognosis of MSA remain limited. To investigate serum NFL’s diagnostic and prognostic value in patients with MSA in a prospective clinical cohort. Two hundred twenty-eight participants were enrolled, including ninety-eight with MSA, seventeen with uncertain MSA at inclusion, fifty-nine with Parkinson’s disease (PD), and fifty-four healthy controls (HCs). Patients with MSA and uncertain diagnoses were followed up. Serum NFL levels were measured with electrochemiluminescence immunoassay at baseline. Patients with MSA and uncertain diagnoses underwent repeated clinical assessments with a median follow-up period of 1.43 years. The final diagnoses included 102 MSA, 62 PD, and 54 HCs. Serum NFL levels were significantly higher in MSA and PD than in HCs. Serum NFL levels, with cutoff values of 223.5 and 218.0 pg/mL, could discriminate between patients with MSA and PD (AUC = 0.930) in the early disease stages, and between MSA-parkinsonism subtypes and PD (AUC = 0.878). Higher serum NFL levels were independently associated with a shorter median time to poor prognosis and death. In addition, reduced levodopa responsiveness was correlated with poor outcomes, and orthostatic hypotension (OH) was linked to a higher risk of death. Serum NFL levels can not only differentiate between MSA, MSA-P, and PD in the early stages of the disease but also serve as a reliable independent predictor of poor prognosis and survival time in MSA.