Analysis of biomarkers in speculative CNS-enriched extracellular vesicles for parkinsonian disorders: a comprehensive systematic review and diagnostic meta-analysis.

BACKGROUND AND OBJECTIVE: Parkinsonian disorders, including Parkinson's disease (PD), multiple system atrophy (MSA), dementia with Lewy bodies (DLB), progressive supranuclear palsy (PSP), and corticobasal syndrome (CBS), exhibit overlapping early-stage symptoms, complicating definitive diagnosis despite heterogeneous cellular and regional pathophysiology. Additionally, the progression and the eventual conversion of prodromal conditions such as REM behavior disorder (RBD) to PD, MSA, or DLB remain challenging to predict. Extracellular vesicles (EVs) are small, membrane-enclosed structures released by cells, playing a vital role in communicating cell-state-specific messages. Due to their ability to cross the blood-brain barrier into the peripheral circulation, measuring biomarkers in blood-isolated speculative CNS enriched EVs has become a popular diagnostic approach. However, replication and independent validation remain challenging in this field. Here, we aimed to evaluate the diagnostic accuracy of speculative CNS-enriched EVs for parkinsonian disorders. METHODS: We conducted a PRISMA-guided systematic review and meta-analysis, covering 18 studies with a total of 1695 patients with PD, 253 with MSA, 21 with DLB, 172 with PSP, 152 with CBS, 189 with RBD, and 1288 HCs, employing either hierarchical bivariate models or univariate models based on study size. RESULTS: Diagnostic accuracy was moderate for differentiating patients with PD from HCs, but revealed high heterogeneity and significant publication bias, suggesting an inflation of the perceived diagnostic effectiveness. The bias observed indicates that studies with non-significant or lower effect sizes were less likely to be published. Although results for differentiating patients with PD from those with MSA or PSP and CBS appeared promising, their validity is limited due to the small number of involved studies coming from the same research group. Despite initial reports, our analyses suggest that using speculative CNS-enriched EV biomarkers may not reliably differentiate patients with MSA from HCs or patients with RBD from HCs, due to their lesser accuracy and substantial variability among the studies, further complicated by substantial publication bias. CONCLUSION: Our findings underscore the moderate, yet unreliable diagnostic accuracy of biomarkers in speculative CNS-enriched EVs in differentiating parkinsonian disorders, highlighting the presence of substantial heterogeneity and significant publication bias. These observations reinforce the need for larger, more standardized, and unbiased studies to validate the utility of these biomarkers but also call for the development of better biomarkers for parkinsonian disorders.

IM-MS and ECD-MS/MS Provide Insight into Modulation of Amyloid Proteins Self-Assembly by Peptides and Small Molecules.

Neurodegenerative proteinopathies are characterized by formation and deposition of misfolded, aggregated proteins in the nervous system leading to neuronal dysfunction and death. It is widely believed that metastable oligomers of the offending proteins, preceding the fibrillar aggregates found in the tissue, are the proximal neurotoxins. There are currently almost no disease-modifying therapies for these diseases despite an active pipeline of preclinical development and clinical trials for over two decades, largely because studying the metastable oligomers and their interaction with potential therapeutics is notoriously difficult. Mass spectrometry (MS) is a powerful analytical tool for structural investigation of proteins, including protein-protein and protein-ligand interactions. Specific MS tools have been useful in determining the composition and conformation of abnormal protein oligomers involved in proteinopathies and the way they interact with drug candidates. Here, we analyze critically the utilization of ion-mobility spectroscopy-MS (IM-MS) and electron-capture dissociation (ECD) MS/MS for analyzing the oligomerization and conformation of multiple amyloidogenic proteins. We also discuss IM-MS investigation of their interaction with two classes of compounds developed by our group over the last two decades: C-terminal fragments derived from the 42-residue form of amyloid ß-protein (Aß42) and molecular tweezers. Finally, we review the utilization of ECD-MS/MS for elucidating the binding sites of the ligands on multiple proteins. These approaches are readily applicable to future studies addressing similar questions and hold promise for facilitating the development of successful disease-modifying drugs against neurodegenerative proteinopathies.

Plasma versus serum for extracellular vesicle (EV) isolation: A duel for reproducibility and accuracy for CNS-originating EVs biomarker analysis.

Blood-derived extracellular vesicles (EVs) are a popular source of biomarkers for central nervous system (CNS) diseases, but inconsistencies in isolation and analysis hinder their clinical translation. This review summarizes recent studies that investigate the impact of different anticoagulated plasma and serum on the yield, purity, and molecular content of EVs. Specifically, the studies compare ethylenediaminetetraacetic acid (EDTA), citrate, heparin plasma, and serum and highlight the risk of contamination from platelet-derived EVs. Here, I offer practical guidelines for standardizing EV isolation and analysis, recommending the use of plasma anticoagulated with acid-citrate-dextrose (ACD) or citrate followed by EDTA and heparin, subgroup analyses for samples from different biobank repositories, and avoiding serum and plasma-to-serum transformation. Other factors like illness, age, gender, meal timing, exercise, circadian timing, and arm pressure during blood draw can alter EV signatures. Yet, how these variables interact with different anticoagulated plasma or serum samples is unclear, necessitating further research. Furthermore, whether the changes are dependent on the isolation or quantification methodology remains an area of investigation. Importantly, the perspective emphasizes the need for consistency in experimental methodologies to improve the reproducibility and clinical applicability of CNS-originating EV biomarker studies. The proposed guidelines, along with ongoing efforts to standardize blood sample handling and collection, may facilitate the development of more reliable and informative CNS-originating EV biomarkers for diagnosis, prognosis, and treatment monitoring of CNS diseases.

Evaluation of α-synuclein in CNS-originating extracellular vesicles for Parkinsonian disorders: A systematic review and meta-analysis.

BACKGROUND & AIMS: Parkinsonian disorders, such as Parkinson's disease (PD), multiple system atrophy (MSA), dementia with Lewy bodies (DLB), progressive supranuclear palsy (PSP) and corticobasal syndrome (CBS), share early motor symptoms but have distinct pathophysiology. As a result, accurate premortem diagnosis is challenging for neurologists, hindering efforts for disease-modifying therapeutic discovery. Extracellular vesicles (EVs) contain cell-state-specific biomolecules and can cross the blood-brain barrier to the peripheral circulation, providing a unique central nervous system (CNS) insight. This meta-analysis evaluated blood-isolated neuronal and oligodendroglial EVs (nEVs and oEVs) α-synuclein levels in Parkinsonian disorders. METHODS: Following PRISMA guidelines, the meta-analysis included 13 studies. An inverse-variance random-effects model quantified effect size (SMD), QUADAS-2 assessed risk of bias and publication bias was evaluated. Demographic and clinical variables were collected for meta-regression. RESULTS: The meta-analysis included 1,565 patients with PD, 206 with MSA, 21 with DLB, 172 with PSP, 152 with CBS and 967 healthy controls (HCs). Findings suggest that combined concentrations of nEVs and oEVs α-syn is higher in patients with PD compared to HCs (SMD = 0.21, p = 0.021), while nEVs α-syn is lower in patients with PSP and CBS compared to patients with PD (SMD = -1.04, p = 0.0017) or HCs (SMD = -0.41, p < 0.001). Additionally, α-syn in nEVs and/or oEVs did not significantly differ in patients with PD vs. MSA, contradicting the literature. Meta-regressions show that demographic and clinical factors were not significant predictors of nEVs or oEVs α-syn concentrations. CONCLUSION: The results highlight the need for standardized procedures and independent validations in biomarker studies and the development of improved biomarkers for distinguishing Parkinsonian disorders.

Biomarkers for parkinsonian disorders in CNS-originating EVs: promise and challenges.

Extracellular vesicles (EVs), including exosomes, microvesicles, and oncosomes, are nano-sized particles enclosed by a lipid bilayer. EVs are released by virtually all eukaryotic cells and have been shown to contribute to intercellular communication by transporting proteins, lipids, and nucleic acids. In the context of neurodegenerative diseases, EVs may carry toxic, misfolded forms of amyloidogenic proteins and facilitate their spread to recipient cells in the central nervous system (CNS). CNS-originating EVs can cross the blood-brain barrier into the bloodstream and may be found in other body fluids, including saliva, tears, and urine. EVs originating in the CNS represent an attractive source of biomarkers for neurodegenerative diseases, because they contain cell- and cell state-specific biological materials. In recent years, multiple papers have reported the use of this strategy for identification and quantitation of biomarkers for neurodegenerative diseases, including Parkinson's disease and atypical parkinsonian disorders. However, certain technical issues have yet to be standardized, such as the best surface markers for isolation of cell type-specific EVs and validating the cellular origin of the EVs. Here, we review recent research using CNS-originating EVs for biomarker studies, primarily in parkinsonian disorders, highlight technical challenges, and propose strategies for overcoming them.

Does the combination of intrathecal fentanyl and morphine improve clinical outcomes in patients undergoing lumbar fusions?

Intrathecal morphine (ITM) has been widely effective in improving postoperative outcomes in patients undergoing a multitude of surgeries, including lumbar spine fusion. A major limitation of ITM administration is the increase in the incidence of respiratory depression in a dose-dependent manner. One way to bypass this is to use a more potent opioid with a shorter half-life, such as fentanyl. This is a retrospective analysis of patients who underwent one- or two-level transforaminal lumbar interbody fusions. The patients received one of two interventions: 0.2mg intrathecal duramorph/morphine (ITM group; n=70), 0.2mg duramorph + 50 mcg fentanyl (ITM + fentanyl group; n=68) and the control group (n=102). Primary outcomes included postoperative pain (Visual Analog Scale) and opioid intake (MED - morphine equivalent dosage, mg) for postoperative days (POD) 1- 4. Secondary outcomes included opioid-related side effects. One-way analyses of variance and follow-up post-hoc Tukey's honest significant difference statistical tests were used to measure treatment effects. Significantly lower POD1 pain scores for both the ITM and ITM + fentanyl groups vs. control were detected, with no difference between the ITM vs. ITM + fentanyl groups. Similar results were found for POD1 MED intake. A multivariate regression analysis controlling for confounding variables did not attenuate the differences seen in POD1 pain scores while revealing that only the ITM + fentanyl predicted a decrease in POD1 MED intake. No differences were seen for postoperative opioid-related side effects. Our study provides support for supplementing a low dose of both intrathecal morphine and fentanyl to improve postoperative outcomes.

Development of a Novel Electrochemiluminescence ELISA for Quantification of α-Synuclein Phosphorylated at Ser129 in Biological Samples.

Synucleinopathies are a group of neurodegenerative diseases including Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). These diseases are characterized by the aggregation and deposition of α-synuclein (α-syn) in Lewy bodies (LBs) in PD and DLB or as glial cytoplasmic inclusions in MSA. In healthy brains, only ∼4% of α-syn is phosphorylated at Ser129 (pS129-α-syn), whereas >90% pS129-α-syn may be found in LBs, suggesting that pS129-α-syn could be a useful biomarker for synucleinopathies. However, a widely available, robust, sensitive, and reproducible method for measuring pS129-α-syn in biological fluids is currently missing. We used Meso Scale Discovery (MSD)'s electrochemiluminescence platform to create a new assay for sensitive detection of pS129-α-syn. We evaluated several combinations of capture and detection antibodies and used semisynthetic pS129-α-syn as a standard for the assay at a concentration range from 0.5 to 6.6 × 104 pg/mL. Using the antibody EP1536Y for capture and an anti-human α-syn antibody (MSD) for detection was the best combination in terms of assay sensitivity, specificity, and reproducibility. We tested the utility of the assay for the detection and quantification of pS129-α-syn in human cerebrospinal fluid, serum, plasma, saliva, and CNS-originating small extracellular vesicles, as well as in mouse brain lysates. Our data suggest that the assay can become a widely used method for detecting pS129-α-syn in biomedical studies including when only a limited volume of sample is available and high sensitivity is required, offering new opportunities for diagnostic biomarkers, monitoring disease progression, and quantifying outcome measures in clinical trials.

Extracellular vesicles from bodily fluids for the accurate diagnosis of Parkinson's disease and related disorders: A systematic review and diagnostic meta-analysis.

Parkinsonian disorders, including Parkinson's disease (PD), multiple system atrophy (MSA), dementia with Lewy body (DLB), corticobasal syndrome (CBS) and progressive supranuclear palsy (PSP) are often misdiagnosed due to overlapping symptoms and the absence of precise biomarkers. Furthermore, there are no current methods to ascertain the progression and conversion of prodromal conditions such as REM behaviour disorder (RBD). Extracellular vesicles (EVs), containing a mixture of biomolecules, have emerged as potential sources for parkinsonian diagnostics. However, inconsistencies in previous studies have left their diagnostic potential unclear. We conducted a meta-analysis, following PRISMA guidelines, to assess the diagnostic accuracy of general EVs isolated from various bodily fluids, including cerebrospinal fluid (CSF), plasma, serum, urine or saliva, in differentiating patients with parkinsonian disorders from healthy controls (HCs). The meta-analysis included 21 studies encompassing 1285 patients with PD, 24 with MSA, 105 with DLB, 99 with PSP, 101 with RBD and 783 HCs. Further analyses were conducted only for patients with PD versus HCs, given the limited number for other comparisons. Using bivariate and hierarchal receiver operating characteristics (HSROC) models, the meta-analysis revealed moderate diagnostic accuracy in distinguishing patients with PD from HCs, with substantial heterogeneity and publication bias. The trim-and-fill method revealed at least two missing studies with null or low diagnostic accuracy. CSF-EVs showed better overall diagnostic accuracy, while plasma-EVs had the lowest performance. General EVs demonstrated higher diagnostic accuracy compared to CNS-originating EVs, which are more time-consuming, labour- and cost-intensive to isolate. In conclusion, while holding promise, utilizing biomarkers in general EVs for PD diagnosis remains unfeasible due to existing challenges. The focus should shift toward harmonizing the field through standardization, collaboration, and rigorous validation. Current efforts by the International Society For Extracellular Vesicles (ISEV) aim to enhance the accuracy and reproducibility of EV-related research through rigor and standardization, aiming to bridge the gap between theory and practical clinical application.

The effect of intraoperative intrathecal opioid administration on the length of stay and postoperative pain control for patients undergoing lumbar interbody fusion.

PURPOSE: In an effort to control postoperative pain more effectively in spinal fusion patients, intraoperative intrathecal morphine (ITM) administration is gaining popularity and acceptance with clinicians. This study seeks to determine the impact of intraoperative intrathecal opioid (ITO) administration following lumbar fusion surgery on postoperative pain and length of hospitalization as primary outcomes. Secondary outcomes will investigate postoperative opioid intake and side effects. METHODS: The retrospective analysis of collected data was performed. The study compared patients undergoing one- or two-level transforaminal interbody fusions between 2019 and 2021 who intraoperatively received two different ITO doses (n = 89) vs. the reference group (n = 48) that did not receive ITO. The patients in the ITO group received either 0.2 mg (n = 44) of duramorph or 0.2 mg duramorph + 50 mcg fentanyl (n = 45). The effect of ITO was evaluated for the first four postoperative days (POD) on pain scores (visual analog scale), length of stay (LOS, hours) and opioid requirement (MED, morphine equivalent dose). RESULTS: In the ITO group, a significant reduction of postoperative pain scores (t(99) = 4.3, p < 0.001) and opioid intake (t(70) = 2.49, p = 0.015) was noted on POD1. Cohen's d effect sizes were 0.76 and 0.50, meaning that postoperative pain and MED intake were reduced by about ¾ to ½ standard deviations (SD) in the ITO group. Further, multivariate regression models revealed that ITO administration predicted lower postoperative pain scores for the two PODs (ß = - 0.83, p < 0.001; ß = - 0.63, p = 0.022) and MED intake for the first two PODs (ß = - 20.8, p = 0.047; ß = - 16.4, p = 0.030). Mean LOS was 15.4 h less in the ITO group (mean ± SD, 63.4 ± 37.1 vs. 78.8 ± 39.6, p = 0.10). CONCLUSIONS: In conclusion, our study provides results in a large sample of patients undergoing transforaminal lumbar fusions. The results demonstrated that ITO administration is effective in reducing POD1 pain scores and POD1-2 opioid requirement while not increasing the risk of any opioid-related side effects.