Vesicle Protein Research Articles

Platelet activation and signaling in thrombus formation.

In thrombosis and hemostasis, the formation of a platelet-fibrin thrombus or clot is a highly controlled process that varies, depending on the pathological context. Major signaling pathways in platelets are well established. However, studies with genetically modified mice have identified the contribution of hundreds of additional platelet-expressed proteins in arterial thrombus formation and bleeding. Using phenotype information of 540 mouse genes, involved in arterial thrombosis and hemostasis, we review current insights into established and novel platelet signaling mechanisms. We discuss pathways involved in platelet adhesion, shape change, integrin activation, intracellular vesicle trafficking and protein processing, granule secretion, aggregate formation and procoagulant activity. Specific attention is paid to the signaling routes used by ITAM-linked, ITIM-linked and G protein-coupled receptors, as well as downstream events feeding into GTPase regulation and protein kinase activation. We further summarize known alterations in platelet responses under conditions of venous, inflammatory and infection-dependent thrombosis, taking into account interactions of platelets with the endothelium, leukocytes and red blood cells. Understanding the genes and proteins involved in platelet signaling in the context of hemostasis, thrombosis and inflammation may lead to improved therapies to prevent and treat thrombotic disorders.

Secretome and Proteome of Extracellular Vesicles Provide Protein Markers of Lung and Colorectal Cancer

Colorectal cancer (CRC) and lung cancer (LC) are leading causes of cancer-related mortality, highlighting the need for minimally invasive diagnostic, prognostic, and predictive markers for these cancers. Proteins secreted by a tumor into the extracellular space directly, known as the tumor secretome, as well as proteins in the extra-cellular vesicles (EVs), represent an attractive source of biomarkers for CRC and LC. We performed proteomic analyses on secretome and EV samples from LC (A549, NCI-H23, NCI-H460) and CRC (Caco2, HCT116, HT-29) cell lines and targeted mass spectrometry on EVs from plasma samples of 20 patients with CRC and 19 healthy controls. A total of 782 proteins were identified across the CRC and LC secretome and EV samples. Of these, 22 and 44 protein markers were significantly elevated in the CRC and LC samples, respectively. Functional annotation revealed enrichment in proteins linked to metastasis and tumor progression for both cancer types. In EVs isolated from the plasma of patients with CRC, ITGB3, HSPA8, TUBA4A, and TLN1 were reduced, whereas FN1, SERPINA1, and CST3 were elevated, compared to healthy controls. These findings support the development of minimally invasive liquid biopsy methods for the detection, prognosis, and treatment monitoring of LC and CRC.

Multicenter study of ovarian cancer score for diagnosing ovarian cancer.

Early detection is crucial for improving survival of patients with ovarian cancer (OC), yet current diagnostic tools lack adequate sensitivity and specificity, especially for early stage disease. The study aimed to validate the serum small extracellular vesicles (sEV) protein based Ovarian Cancer Score (OCS) in detecting OC. This multicenter study included 1183 adult females with adnexal masses from four hospitals in China (October 2019-April 2023). Of these, 1024 samples were prospectively collected, and 159 were from biobanks. All serum samples were collected before surgery. The concentrations of sEV carbohydrate antigen 125 (CA125), human epididymis protein 4 (HE4), and complement component 5a protein (C5a) were quantified using chemiluminescence immunoassay and then used for calculating OCS. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated. The OCS demonstrated high sensitivity (95.4%) and specificity (90.4%) in diagnosis of OC in the prospective cohort (n=1024) and in total cases (n=1183, 95.5% and 90.2%), with stable performance across menopausal status and FIGO stages. The OCS maintained a high specificity in premenopausal patients (89.6%) and postmenopausal patients (92.1%). The OCS showed high sensitivity in early stage epithelial OC (FIGO I: 89.7%, I+II: 91.4%), in patients aged ≤45years (92.7%), and in patients with normal CA125 levels (72.7%), although these results were obtained from subgroups with small sample sizes. This multicenter study demonstrated that the OCS is a promising non-invasive diagnostic tool for the detection of OC. This study was registered at ClinicalTrials.gov: NCT06366997.

Integrative analysis of miRNAs and proteins in plasma extracellular vesicles of patients with familial hypercholesterolemia.

Familial Hypercholesterolemia (FH) is a monogenic disease that leads to early-onset atherosclerosis. Causative mutations in FH-related genes are found in 60-80 % of patients, while epigenetic factors may contribute to mutation-negative cases. This study analyzed miRNAs and proteins from plasma-derived extracellular vesicles (EVs) of FH patients to explore their contribution in FH diagnosis. Clinical and laboratory data were obtained from 54 FH patients and 38 normolipidemic individuals. FH-related gene variants were identified using exon-targeted gene sequencing. Plasma EVs miRNome and proteome were analysed using small RNA sequencing and liquid chromatography/mass spectrometry. Thirteen FH patients carried LDLR deleterious variants (MD group), while 41 did not (non-MD group). Over 2000 miRNAs were detected in plasma EVs, with miR-122-5p higher in FH patients compared to controls, and miR-21-5p higher in the MD group than in the non-MD group (p < 0.05). Proteomic analysis identified 300 proteins with 18 out of 38 proteins more abundant in EVs than in total plasma. Eighteen EVs-derived proteins had differential abundance in FH patients compared to control group (p < 0.05). EV levels of miR-122-5p, miR-21-5p and 12 proteins were correlated with serum lipids (p < 0.05). The integrative analysis between dysregulated miRNAs (miR-122-5p and miR-21-5p) and altered proteins (APOD, APOF, MBL2 and MASP1) from EVs identified several common pathways involved in cholesterol metabolism. Co-regulation of plasma EVs miR-122-5p, miR-21-5p, APOD, APOF, MBL2 and MASP1 and their correlation with serum lipids suggest their involvement in impaired cholesterol metabolism and may be useful as biomarkers of FH severity.

Molecular Dynamics Simulations of the SNARE Complex Interacting with Synaptotagmin, Complexin, and Lipid Bilayers.

Molecular dynamics (MD) simulations enable in silico investigation of the dynamic behavior of proteins and protein complexes. Here, we describe MD simulations of the SNARE bundle forming the complex with the neuronal proteins Synaptotagmin-1 (Syt1) and Complexin (Cpx). Syt1 is the synaptic vesicle (SV) protein that serves as the neuronal calcium sensor and triggers synaptic fusion upon calcium binding, and this process is promoted and accelerated by Cpx. The fusion depends on the Syt1 interactions with the SNARE-Cpx complex and with the lipid bilayer of the presynaptic membrane (PM). The MD simulations of the PM-Syt1-SNARE-Cpx-SV molecular system described here enabled us to investigate how this protein-lipid complex promotes the merging of SV and PM, triggering synaptic fusion.

Control of Synaptotagmin-1 Trafficking by SV2A-Mechanism and Consequences for Presynaptic Function and Dysfunction.

Synaptic vesicle protein 2A (SV2A) is an abundant synaptic vesicle cargo with an as yet unconfirmed role in presynaptic function. It is also heavily implicated in epilepsy, firstly being the target of the leading anti-seizure medication levetiracetam and secondly with loss of function mutations culminating in human disease. A range of potential presynaptic functions have been proposed for SV2A; however its interaction with the calcium sensor for synchronous neurotransmitter release, synaptotagmin-1 (Syt1), has received particular attention over the past decade. In this review we will assess the evidence that the primary role of SV2A is to control the expression and localisation of Syt1 at the presynapse. This will integrate biochemical, cell biological and physiological studies where the interaction, trafficking and functional output of Syt1 is altered by SV2A. The potential for SV2A-dependent epilepsy to be a result of dysfunctional Syt1 expression and localisation is also discussed. Finally, a series of key open questions will be posed that require resolution before a definitive role for SV2A in Syt1 function in health and disease can be confirmed.

Upregulation of rod ribbon synapse components implies homeostatic presynaptic scaling in early‐stage retinitis pigmentosa

Aims/Purpose: Photoreceptor death triggers retinal rewiring and remodeling that are generally thought to be detrimental for vision. Some recent research results challenge this dogma. It was recently shown that in early‐stage retinitis pigmentosa (RP), the retina compensates for the reduced rod population activity by upscaling synaptic transmission between the remaining rods and rod bipolar cells. The purpose of the current project is to study the molecular mechanism behind this phenomenon.Methods: To model the primary pathology, P23H mouse model of autosomal dominant RP was used. Notably, wild‐type mice were not used as controls, instead, cone‐transducin (GNAT2) knockout mice that lack cone photoreceptor functionality were used. To produce “rod‐function‐only” RP mice, P23H and Gnat2‐/‐ mice were cross‐bred. Single cell RNA‐sequencing (scRNA‐seq; Gnat2‐/‐, n = 4; P23H/Gnat2‐/‐, n = 4) and retinal global proteomics (Gnat2‐/‐, n = 5; P23H/Gnat2‐/‐, n = 10) analyses were performed at P30. Statistical significance was set at q &lt; 0.05. Additional protein expression analyses for synaptic proteins were performed using immunohistochemistry and immunoblotting.Results: ScRNA‐seq analysis revealed upregulation of several major ribbon synapse genes exclusively in P23H/Gnat2‐/‐ mouse rods. These included genes that are crucial in presynaptic structure and neurotransmitter release, such as the Ca2+ sensor Syt1, snare protein complex component Snap25, ribbon protein Ctbp2, and the synaptic vesicle protein Sv2b. In addition, upregulation of the glutamate transporter Slc1a2 was found. Based on proteomics data, STX1B and SYT7 were overexpressed in whole P23H/Gnat2‐/‐ mouse retinas as compared to controls.Conclusions: Early‐stage RP in mice associates with increased expression of several rod ribbon synapse components that play a role in glutamate release. These results imply that regulation of presynaptic glutamate release may play a role in improving retinal synaptic transmission during RP.

Protein profile of purified plasma- and aqueous humor-derived extracellular vesicles from patients with ocular toxoplasmosis.

To characterize the extracellular vesicle protein cargo in the aqueous humor and plasma of patients with ocular toxoplasmosis. Aqueous humor and plasma were collected from six patients with active ocular toxoplasmosis and six patients with cataract. Extracellular vesicles were isolated, and western blotting and mass spectrometry were performed for protein analysis. All plasma samples from patients with ocular toxoplasmosis and cataract were positive for the tetraspanins CD63 and TSG101. However, the aqueous humor from patients with ocular toxoplasmosis was positive only for CD63. Sixty-seven new unreported proteins were identified in the aqueous humor and plasma of patients with the ocular toxoplasmosis and cataract. Of the 67 proteins, 10 and 7 were found only in the cataract and ocular toxoplasmosis groups, respectively. In general, these proteins were involved in immune system activation and retina homeostasis and were related to infections and retina-associated diseases. The distinct protein signatures between ocular toxoplasmosis and cataract may be helpful in the differential diagnosis of ocular toxoplasmosis. However, more studies are needed to better understand the role of these proteins in the pathogenesis of ocular toxoplasmosis.

Circulating large extracellular vesicles as diagnostic biomarkers of indeterminate thyroid nodules: multi-platform omics analysis.

While most thyroid nodules are benign, 7-15% are malignant. Patients with indeterminate thyroid nodules (specifically Bethesda IV/Thy3f) often undergo diagnostic hemithyroidectomy to reach a diagnosis on final histology. The aim of this study was to assess the feasibility of circulating large extracellular vesicles as diagnostic biomarkers in patients presenting with Thy3f thyroid nodules. This was a two-gate diagnostic accuracy study; patients with Thy3f thyroid nodules were age, sex and body mass index matched to healthy individuals. Final histology confirmed benign and malignant diagnoses. Plasma large extracellular vesicle counts were quantified using flow cytometry. Large extracellular vesicle microRNA and protein profiles were identified using next generation sequencing and mass spectrometry, respectively. A total of 42 patients with Thy3f nodules (22 with cancer, 20 with non-cancer diagnosis) and 16 healthy controls were included. Total large extracellular vesicle concentrations and the concentrations of extracellular vesicles expressing epithelial cell adhesion molecule and the cancer markers atypical chemokine receptor type 7, extracellular matrix metalloproteinase inducer and syndecan-4 were significantly higher in patients with Thy3f nodules (cancer and non-cancer) compared with healthy individuals. In patients with cancerous versus non-cancer Thy3f nodules, one microRNA was upregulated: mir-195-3p (P < 0.001). Five were downregulated: mir-3176 (P < 0.001), mir-205-5p (P < 0.001), novel-hsa-mir-208-3p (P < 0.001), mir-3529-3p (P = 0.01) and let-7i-3p (P = 0.02). Furthermore, three large extracellular vesicle proteins (kallikrein-related peptidase11 (KLK11) (P = 0.001), alpha-1-acid glycoprotein 2 (A1AG2) (P <0.001) and small integral membrane protein 1 (SMIM1) (P = 0.04)) were significantly upregulated, while 20 large extracellular vesicle proteins were significantly downregulated (most downregulated: chemokine (C-X-C motif) ligand 7 (CXCL7), tubulin beta chain 1 (TBB1), binding immunoglobulin protein (BIP) and actinin alpha 1 (ACTN1) (P < 0.001)) in cancerous compared with non-cancer Thy3f nodules. Circulating large extracellular vesicle miRNA and protein profiles have a high diagnostic value to discriminate between benign and malignant nodules for patients with Thy3f cytology. Further validation for clinical performance will be needed.

Longitudinal synaptic loss versus tau Braak staging in amnestic mild cognitive impairment.

The longitudinal progression of synaptic loss in Alzheimer's disease (AD) and how it is affected by tau pathology remains poorly understood. Thirty patients with amnestic mild cognitive impairment (aMCI) and 26 healthy controls underwent cognitive evaluations and tau, synaptic vesicle protein 2A (SV2A), and amyloid positron emission tomography. Twenty-one aMCI underwent 2-year follow-up (FU) investigations. Tau levels in aMCI increased longitudinally in Braak regions III through VI but not in Braak regions I and II. SV2A decreased longitudinally in all Braak regions in aMCI. Baseline tau was negatively associated with longitudinal SV2A loss in early Braak regions and with SV2A at FU across regions. Baseline tau and longitudinal change in SV2A were associated with longitudinal cognitive decline. Tau accumulation reaches a plateau in early Braak regions already in the aMCI stage of AD. In early Braak regions, the association between baseline tau and longitudinal SV2A loss might reflect synaptic dysfunction caused by tau pathology. Tau accumulation reached a plateau in early Braak regions in amnestic mild cognitive impairment (aMCI) patients. aMCI patients show widespread longitudinal decrease in synaptic vesicle protein 2A (SV2A) over 2 years. Baseline tau was predictive for longitudinal SV2A loss. The tau-SV2A relation showed individual variability and was negative across patients. Baseline tau and longitudinal SV2A change were associated with change in cognition.

Effects of Different Processing on miRNA and Protein in Small Extracellular Vesicles of Goat Dairy Products.

Small extracellular vesicles (sEVs) are nanosized vesicles with biological activities that exist in milk, playing functional roles in immunity, gut balance, and the nervous system. Currently, little is known about the impact of processing on milk sEVs. In this study, sEVs were collected from raw goat milk (g-sEV), pasteurized goat milk (pg-sEV), and goat milk powder (p-sEV) using a sucrose cushion centrifugation combined with qEV chromatography. Then, the sEVs were identified and compared using NTA, Western blot, and TEM. After extracting RNA and the total proteome from sEVs derived from different samples, the RNA was subjected to high-throughput sequencing, and peptide fragments were analyzed using mass spectrometry. Finally, GO and KEGG pathway analyses were performed on the results. The characterization results revealed a decrease in diameter as the level of processing increased. High-throughput sequencing results showed that all three types of small extracellular vesicles were found to be rich in miRNA, and no significant differences were observed in the most abundant sEV species. Comparing with g-sEV, there were 3938 and 4645 differentially expressed miRNAs in pg-sEV and p-sEV, respectively, with the majority of them (3837 and 3635) being downregulated. These differentially expressed miRNAs were found to affect biological processes or signaling pathways such as neurodevelopment, embryonic development, and transcription. Proteomic analysis showed that there were 339 differentially expressed proteins between g-sEV and pg-sEV, with 209 proteins being downregulated. Additionally, there were 425 differentially expressed proteins between g-sEV and p-sEV, with 293 proteins being downregulated. However, no significant differences were observed in the most abundant protein species among the three types of sEVs. Enrichment analysis indicated that the differentially expressed proteins were associated with inflammation, immunity, and other related processes. These results indicate that extracellular vesicles have a protective effect on their cargo, while processing steps can have an impact on the size and quantity of the sEVs. Furthermore, processing can also lead to the loss of immune-related miRNA and proteins in sEVs.

Post mortem validation and mechanistic study of UCB-J in progressive supranuclear palsy patients' brains.

Progressive supranuclear palsy (PSP) is a devastating 4R tauopathy affecting motor functions and is often misdiagnosed/underdiagnosed due to a lack of specific biomarkers. Synaptic loss is an eminent feature of tauopathies including PSP. Novel synaptic positron emission tomography tracer UCB-J holds great potential for early diagnosis; however, there is a substantial knowledge gap in terms of the mechanism and the extent and nature of synaptic loss in PSP. Here, we report an in-depth post mortem validation and mechanistic study of UCB-J in PSP and control brains using radioligand/autoradiography binding studies, alongside biochemical correlation analyses of synaptic markers. 3H-UCB-J targeted synaptic vesicle protein 2A protein with high specificity and demonstrated a distinct interrelation with synaptic markers in PSP patients' brain regions. The loss of UCB-J binding in the early and severely affected globus pallidus of PSP patients' brains revealed deficits of glutamate/GABAergic synaptic terminals. Cortical and subcortical 4R tau load differentially impacted synaptic marker profiles across PSP patients, warranting further investigation. UCB-J targeted synaptic vesicle protein 2A with high specificity in progressive supranuclear palsy (PSP) brains and demonstrated a conserved single nM binding site across different brain regions. UCB-J depicted prominent synaptic loss at the synaptosome levels and revealed deficits of glutamate/GABAergic synaptic terminals in the early affected globus pallidus of PSP brains as compared to the control. Cortical and subcortical 4R tau load distinctly influenced synaptic markers profile across PSP patients and highlighted that presynaptic "ubiquitous" markers individually might not be able to represent the complete state of synaptic deficits/loss in PSP brains.

The cellular and extracellular proteomic signature of human dopaminergic neurons carrying the LRRK2 G2019S mutation.

Extracellular vesicles are easily accessible in various biofluids and allow the assessment of disease-related changes in the proteome. This has made them a promising target for biomarker studies, especially in the field of neurodegeneration where access to diseased tissue is very limited. Genetic variants in the LRRK2 gene have been linked to both familial and sporadic forms of Parkinson's disease. With LRRK2 inhibitors entering clinical trials, there is an unmet need for biomarkers that reflect LRRK2-specific pathology and target engagement. In this study, we used induced pluripotent stem cells derived from a patient with Parkinson's disease carrying the LRRK2 G2019S mutation and an isogenic gene-corrected control to generate human dopaminergic neurons. We isolated extracellular vesicles and neuronal cell lysates and characterized their proteomic signature using data-independent acquisition proteomics. Then, we performed differential expression analysis to identify dysregulated proteins in the mutated line. We used Metascape and gene ontology enrichment analysis on the dysregulated proteomes to identify changes in associated functional networks. We identified 595 significantly differentially regulated proteins in extracellular vesicles and 3,205 in cell lysates. We visualized functionally relevant protein-protein interaction networks and identified key regulators within the dysregulated proteomes. Using gene ontology, we found a close association with biological processes relevant to neurodegeneration and Parkinson's disease. Finally, we focused on proteins that were dysregulated in both the extracellular and cellular proteomes. We provide a list of ten biomarker candidates that are functionally relevant to neurodegeneration and linked to LRRK2-associated pathology, for example, the sonic hedgehog signaling molecule, a protein that has tightly been linked to LRRK2-related disruption of cilia function. In conclusion, we characterized the cellular and extracellular proteome of dopaminergic neurons carrying the LRRK2 G2019S mutation and proposed an experimentally based list of biomarker candidates for future studies.

Single‐Cell Secretome Profiling Enabled by Selective Enrichment and NanoLC‐MS/MS Analysis

AbstractRecent advances in single‐cell proteomics enable the direct profiling of thousands of proteins from a single mammalian cell. However, due to the bottlenecks in detecting low‐abundance secreted proteins and extracellular vesicle (EV) proteins (collectively referred to as the secretome) against a background of high‐abundance proteins in serum‐containing culture medium, the comprehensive investigation of the secretome at the single‐cell level using nanoLC‐MS/MS still remains challenging. Herein, we report a novel single‐cell secretome profiling (SCSP) method by integrating the metabolic labeling of newly synthesized proteins, click chemistry‐based enrichment, and in situ digestion of the labeled secretome in an alkyne‐functionalized capillary micro‐reactor, followed by nanoLC‐MS/MS analysis. By this method, an average of 389 protein groups were quantified from the secretome of single HeLa cells (n=17), with a total of 752 protein groups confidently identified in the single‐cell secretome, which is a significant increase compared to the previously reported targeted analysis limited to dozens of secreted proteins by antibody recognition. These results indicated that our developed SCSP method would provide a powerful tool to gain insights into secretion heterogeneity and intercellular communication at the single‐cell level.

Single-Cell Secretome Profiling Enabled by Selective Enrichment and NanoLC-MS/MS Analysis.

Recent advances in single-cell proteomics enable the direct profiling of thousands of proteins from a single mammalian cell. However, due to the bottlenecks in detecting low-abundance secreted proteins and extracellular vesicle (EV) proteins (collectively referred to as the secretome) against a background of high-abundance proteins in serum-containing culture medium, the comprehensive investigation of the secretome at the single-cell level using nanoLC-MS/MS still remains challenging. Herein, we report a novel single-cell secretome profiling (SCSP) method by integrating the metabolic labeling of newly synthesized proteins, click chemistry-based enrichment, and in situ digestion of the labeled secretome in an alkyne-functionalized capillary micro-reactor, followed by nanoLC-MS/MS analysis. By this method, an average of 389 protein groups were quantified from the secretome of single HeLa cells (n=17), with a total of 752 protein groups confidently identified in the single-cell secretome, which is a significant increase compared to the previously reported targeted analysis limited to dozens of secreted proteins by antibody recognition. These results indicated that our developed SCSP method would provide a powerful tool to gain insights into secretion heterogeneity and intercellular communication at the single-cell level.

Morphological features of synaptic structures associated with amyloid plaques in the human cerebral cortex

BACKGROUND: Synapse damage and loss are fundamental to the pathogenesis of Alzheimer’s disease (AD) and lead to reduced cognitive function. Therefore, the study of synaptic abnormalities is crucial for understanding the pathogenesis and progression of AD. Synaptophysin, a transmembrane protein of synaptic vesicles, is most widely used as an immunohistochemical marker of synapses. AIM: to study the morphological features of synaptophysin–containing elements in the human cerebral cortex with amyloid plaques. METHODS: This study was made on the cerebral cortex samples of men and women aged 65 to 94 years (n = 10). Synapses and amyloid plaques were simultaneously detected with a new original light microscopy technique. This technique is based on immunohistochemical staining for synaptophysin and histochemical staining of amyloid plaques with Alcian blue. RESULTS: Abnormal synaptophysin-immunopositive structures presumed to be dystrophic presynapses were found within most of the identified amyloid plaques. These structures have large sizes and indistinct shapes and are present exclusively within amyloid plaques. Interestingly, corpora amylacea were also identified in all samples. They have spherical shapes and are located mainly in the perivascular, periventricular, and subpial regions of the brain. In all analyzed samples, synaptophysin-immunopositive terminals surrounding corpora amylacea had normal morphology and density and showed no signs of pathology. CONCLUSION: In this work, abnormal synaptophysin-containing structures associated with amyloid plaques were found in the human cerebral cortex using the original staining technique. Further study of these structures holds promise for identifying new biomarkers of synaptic disorganization, including in Alzheimer's disease.

Hyperactive neuronal networks facilitate tau spread in an Alzheimer's disease mouse model.

Pathological tau spreads throughout the brain along neuronal connections in Alzheimer's disease (AD), but the mechanisms that underlie this process are poorly understood. Given the high incidence and deleterious consequences of epileptiform activity in AD, we hypothesized neuronal hyperactivity and seizures are key factors in tau spread. To examine these interactions, we created a novel mouse model involving the cross of targeted recombination in active populations (TRAP) mice and the 5 times familial AD (5XFAD; 5X-TRAP) model allowing for the permanent fluorescent labelling of neuronal activity. To establish a causal role of seizures in tau spread, we seeded mice with human AD brain-derived tau lysate and induced seizures with pentylenetetrazol (PTZ) kindling. Comprehensive brain mapping of tau pathology and neuronal activity revealed that basal hyperactivity in 5X-TRAP mice was associated with increased tau spread, which was exacerbated by seizure induction through activated networks and correlated with memory deficits. Computational modeling revealed that anterograde tau spread was elevated in 5X-TRAP mice and that regional neuronal activity was predictive of tau spread to that brain region. On a cellular level, we found that in both saline and PTZ-treated 5X-TRAP mice, hyperactive neurons disproportionately contributed to the spread of tau. Further, we found that Synaptogyrin-3, a synaptic vesicle protein that interacts with tau, was increased following PTZ kindling in 5X-TRAP mice, possibly indicative of a synaptic mechanism underlying seizure-exacerbated tau spread. Importantly, postmortem AD brain tissue from patients with a history of seizures showed increased tau pathology in patterns indicative of increased spread and increased Synaptogyrin-3 levels compared to those without seizures. Overall, our study identifies neuronal hyperactivity and seizures as key factors underlying the pathobiological and cognitive progression of AD. Therapies targeting these factors should be tested clinically to slow tau spread and AD progression.

Challenges of MS-based small extracellular vesicles proteomics.

Proteomic profiling of small extracellular vesicles (sEV) is a powerful tool for discovering biomarkers of various diseases. This process most often assisted by mass spectrometry (MS) usually lacks standardization and recognition of challenges which may lead to unreliable results. General recommendations for sEV MS analyses have been briefly given in the MISEV2023 guidelines. The present work goes into detail for every step of sEV protein profiling with an overview of factors influencing such analyses. This includes reporting and defining the sEV source and vesicle isolation, protein solubilization and digestion, 'offline' and 'online' sample complexity reduction, the analysis type itself, and subsequent data analysis. Every stage in this process affects the others, which could result in different outcomes. Although characterization and comparisons of different sEV isolation methods are known and accessible and MS-based profiling details are provided for cell or tissue samples, no consensus work has been ever published to describe the whole process of sEV proteomic analysis. Reliable results can be obtained from sEV profiling provided that the analysis is well planned, prepared for, and backed by pilot studies or appropriate research.

Plasma Extracellular Vesicle-Associated Proteins as Promising Diagnostic Biomarkers of Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) is a significant factor causing blindness in adults. However, the clinical diagnosis of AMD is relatively challenging, due to the shortcomings of the existing clinical examination methods and the latent period of retinal damage before macular degeneration becomes apparent. This study aims to explore the potential of extracellular vesicles (EVs) protein chips for early diagnosis of AMD using patients' plasma samples. To achieve early diagnosis of AMD, this study utilized a high-throughput platform for liquid biopsy based on EVs protein chips. Forty AMD patients and 41 normal individuals were recruited. Through machine learning methods, we identified that ATP-binding cassette transporter A1 (ABCA1) is an EVs protein marker for diagnosing AMD. Additionally, a validation set was constructed using the random forest method for verification. The results of the study indicated that ABCA1 is a reliable biomarker for diagnosing AMD. The validation using the random forest method confirmed the robustness and reliability of ABCA1 as a diagnostic marker. This finding suggested that ABCA1 can serve as a new promising liquid biopsy-based marker for diagnosing macular degeneration. The utilization of EVs protein chips, combined with machine learning methods, can effectively identify ABCA1 as a biomarker for the early diagnosis of AMD. This approach offers a promising new method for liquid biopsy diagnostics, potentially improving the clinical diagnosis and management of macular degeneration.

Exposure to an environmentally representative mixture of polybrominated diphenyl ethers (PBDEs) alters zebrafish neuromuscular development

Polybrominated diphenyl ethers (PBDEs) are a prevalent group of brominated flame retardants (BFRs) added to several products such as electronics, plastics, and textiles to reduce their flammability. They are reported as endocrine disruptors and neurodevelopmental toxicants that can accumulate in human and wildlife tissues, thus making their ability to leach out of products into the environment a great cause for concern. In this study, zebrafish (Danio rerio) embryos and larvae were exposed to a wide concentration range (1.5, 15, 150 and 300 pM) of a PBDE mixture from one to six days post-fertilization (dpf). Hatching rates, mortality and general morphology were assessed during the exposure period. A delay in hatching was observed at the two highest PBDEs concentrations and mortality rate increased at 6 dpf. By 4 dpf, larvae exposed to 150 pM and 300 pM PBDEs developed an upcurved phenotype. Analysis of motor behavior at 6 dpf revealed that PBDE exposure acutely reduced locomotion. To further analyze these motor deficits, we assessed the neural network density and motor neuron and neuromuscular junctions (NMJ) development by immunostaining and imaging. Acetylated α-tubulin staining revealed a significant loss of neurons in a dose-dependent manner. Synaptic vesicle protein 2 (SV2) and ⍺-bungarotoxin (⍺-BTX) staining revealed a similar pattern, with a significant loss of SV2 and nicotinic acetylcholine receptors, thus preventing the colocalization of presynaptic neurons with postsynaptic neurons. Consistent with these results, the presence of cleaved caspase-3 and acridine orange positive cells showed increased cell death in zebrafish larvae exposed to PBDEs. Our results suggest that exposure to PBDEs leads to deficits in the zebrafish neuromuscular system through neuron death, inducing morphological and motor deficiencies throughout their development. They provide valuable insight into the neurotoxic effects of PBDEs, further highlighting the relevance of the zebrafish model in toxicological studies.