Biology subjects

Gilboa, T.

Publications and source records attributed to Gilboa, T..

5 recordsLinked to original sources

bioRxiv2026

Single-Molecule Imaging Reveals Differential Stability of Alpha-Synuclein Aggregates

-Synuclein (-syn) aggregation is central to Parkinsons disease (PD), yet measurements in biofluids are confounded by the coexistence of monomeric and aggregated species. Using Syn-IMAGR, a single-molecule imaging platform with sub-femtomolar sensitivity, we show that purified -syn aggregates undergo dilution-induced disassembly, revealing a concentration-dependent equilibrium. Applied to postmortem brain lysates, Syn-IMAGR distinguishes physiological -syn multimers, which are dimmer and readily dissociate upon dilution, from PD-associated aggregates, which remain detectable and exhibit greater structural resistance to disruption. These results indicate that -syn assemblies occupy distinct stability regimes, with PD-associated aggregates representing a more persistent and less dilution-sensitive structural state. Syn-IMAGR thus provides a quantitative framework for resolving -syn species and for probing their concentration-dependent equilibrium.

neuroscience↗

bioRxiv2024

Identification of markers for the isolation of neuron-specific extracellular vesicles

Extracellular vesicles (EVs) are released by all cells and contain RNA and protein from their cell of origin. EVs in biofluids could be used as diagnostic biomarkers to non-invasively report the state of inaccessible cells, such as neurons in the brain. As biofluids such as cerebrospinal fluid (CSF) and plasma contain EVs originating from many different cells, isolating cell type-specific EVs and measuring their cargo could help determine the state of specific cell types. Here, we demonstrate an approach aiming to immuno-isolate EVs from neurons based on neuron-derived protein surface markers. We first developed a framework to select transmembrane proteins suitable as neuron-specific EV markers based on gene expression and EV proteomics data. Leveraging a novel, high-purity EV isolation method we developed, we further cataloged the proteins present on EVs in human CSF and plasma. Using ultrasensitive immunoassays against several of the predicted neuron-specific proteins, we confirmed one marker, NRXN3 as present on EVs in CSF and plasma by size exclusion chromatography (SEC) and density gradient centrifugation (DGC). Finally, we developed efficient EV immuno-isolation methods and applied them to isolate NRXN3+ EVs. Our study provides a general methodology for the isolation of cell-type specific EVs and paves the way for the use of neuron-derived EVs to study and diagnose neurological disease.

molecular biology↗

bioRxiv2023

Assessment of extracellular vesicle protein cargo as neurodegenerative disease biomarkers

Extracellular vesicles (EVs) are released by all cells and hold great promise as a class of biomarkers. As EVs represent a way of capturing molecular information about the proteins inside of cells, EVs from biofluids could be used to better understand and diagnose disease from difficult to access organs such as the brain. This promise has led to increased interest in measuring EV proteins from both total EVs as well as brain-derived EVs isolated from the blood. However, the measurement of cargo proteins in EVs has been challenging because EVs are present at low levels and EV isolation methods are imperfect at separating EVs from free proteins. Thus, it is difficult to know whether a protein measured after EV isolation is truly inside EVs. In this study, we developed methods to measure whether a protein is inside EVs and quantify the ratio of a protein in EVs relative to total plasma. To achieve this, we combined a high-yield size exclusion chromatography (SEC) protocol with an optimized protease protection assay and Single Molecule Array (Simoa) digital ELISA assays for ultrasensitive measurement of proteins inside EVs. We applied these methods to analyze key proteins involved in neurodegenerative diseases: -synuclein, Tau, A{beta}40, and A{beta}42. We found that -synuclein and Tau are present in plasma EVs at a small fraction of the levels in total plasma, whereas A{beta}40 and A{beta}42 are undetectable in plasma EVs. This work provides a framework for determining the levels of proteins in EVs and represents an important step in the development of EV diagnostics for diseases of the brain, as well as other organs.

neuroscience↗

bioRxiv2023

Improved Isolation of Extracellular Vesicles by Removal of Both Free Proteins and Lipoproteins

Extracellular vesicles (EVs) are released by all cells into biofluids such as plasma. The separation of EVs from highly abundant free proteins and similarly-sized lipoproteins remains technically challenging. We developed a digital ELISA assay based on Single Molecule Array (Simoa) technology for ApoB-100, the protein component of several lipoproteins. Combining this ApoB-100 assay with previously developed Simoa assays for albumin and three tetraspanin proteins found on EVs (Ter-Ovanesyan*, Norman* et al., 2021), we were able to measure the separation of EVs from both lipoproteins and free proteins. We used these five assays to compare EV separation from lipoproteins using size exclusion chromatography (SEC) with resins containing different pore sizes. We also developed improved methods for EV isolation based on combining several types of chromatography resins in the same column. We present a simple approach to quantitatively measure the main contaminants of EV isolation in plasma and apply this approach to develop novel methods for isolating highly-pure EVs from human plasma. These methods will enable applications where high purity EVs are required to both understand EV biology and profile EVs for biomarker discovery.

bioengineering↗

bioRxiv2021

Protective heterologous T cell immunity in COVID-19 induced by MMR and Tdap vaccine antigens

T cells are critical for control of viral infection and effective vaccination. We investigated whether prior Measles-Mumps-Rubella (MMR) or Tetanus-Diphtheria-pertussis (Tdap) vaccination elicit cross-reactive T cells that mitigate COVID-19. Using co-cultures of antigen presenting cells (APC) loaded with antigens and autologous T cells, we found a high correlation between responses to SARS-CoV-2 (Spike-S1 and Nucleocapsid) and MMR and Tdap vaccine proteins in both SARS-CoV-2 infected individuals and individuals immunized with mRNA-based SARS-CoV-2 vaccines. The overlapping T cell population contained effector memory T cells (TEMRA) previously implicated in anti-viral immunity and their activation required APC-derived IL-15. TCR- and scRNA-sequencing detected cross-reactive clones with TEMRA features among the cells recognizing SARS-CoV-2, MMR and Tdap epitopes. A propensity-weighted analysis of 73,582 COVID-19 patients revealed that severe disease outcomes (hospitalization and transfer to intensive care unit or death) were reduced in MMR or Tdap vaccinated individuals by 38-32% and 23-20% respectively. In summary, SARS-CoV-2 re-activates memory T cells generated by Tdap and MMR vaccines, which may reduce disease severity.

immunology↗