Biology subjects

Whiteman, S.

Publications and source records attributed to Whiteman, S..

4 recordsLinked to original sources

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↗

bioRxiv2024

Instability of excitatory synapses in experimental autoimmune encephalomyelitis and the outcome for excitatory circuit inputs to individual cortical neurons

Synapses are lost on a massive scale in the brain and spinal cord of people living with multiple sclerosis (PwMS), and this synaptic loss extends far beyond demyelinating lesions. Post-mortem studies show the long-term consequences of multiple sclerosis (MS) on synapses but do not inform on the early impacts of neuroinflammation on synapses that subsequently lead to synapse loss. How excitatory circuit inputs are altered across the dendritic tree of individual neurons under neuroinflammatory stress is not well understood. Here, we directly assessed the structural dynamics of labeled excitatory synapses in experimental autoimmune encephalomyelitis (EAE) as a model of immune-mediated cortical neuronal damage. We used in vivo two-photon imaging and a synthetic tissue-hydrogel super-resolution imaging technique to reveal the dynamics of excitatory synapses, map their location across the dendritic tree of individual neurons, and examine neurons at super-resolution for synaptic loss. We found that excitatory synapses are destabilized but not lost from dendritic spines in EAE, starting with the earliest imaging session before symptom onset. This led to dramatic changes in excitatory circuit inputs to individual cells. In EAE, stable synapses are replaced by synapses that appear or disappear across the imaging sessions or repeatedly change at the same location. These unstable excitatory inputs occur closer to one another in EAE than in healthy controls and are distributed across the dendritic tree. When imaged at super-resolution, we found that a small proportion of dendritic protrusions lost their presynapse and/or postsynapse. Our finding of diffuse destabilizing effects of neuroinflammation on excitatory synapses across cortical neurons may have significant functional consequences since normal dendritic spine dynamics and clustering are essential for learning and memory.

neuroscience↗

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↗