size-exclusion-chromatography-for-separating-extracellular-vesicles

Size Exclusion Chromatography for Separating Extracellular Vesicles from Conditioned Cell Culture Media

Extracellular vesicles (EVs) are nano-sized lipid-membrane bound structures that are released from all cells, are present in all biofluids, and contain ...

Penn State Erie, The Behrend College

Magee Womens Research Institute—Allied Member

Alzheimer's disease (AD) and other neurodegenerative diseases are characterized by chronic neuroinflammation and a reduction in brain energy metabolism. An important role has emerged for small, non-coding RNA molecules known as microRNAs (miRNAs) in the pathophysiology of many neurodegenerative disorders. As epigenetic regulators, miRNAs possess the capacity to regulate and fine tune protein production by inhibiting translation. Several miRNAs, which include miR-146a, are elevated in the brain, CSF, and plasma of AD patients. miR-146a participates in pathways that regulate immune activation and has several mRNA targets which encode for proteins involved in cellular energy metabolism. An additional role for extracellular vesicles (EVs) has also emerged in the progression AD, as EVs can transfer functionally active proteins and RNAs from diseased to healthy cells. In the current study, we exposed various cell types present within the CNS to immunomodulatory molecules and observed significant upregulation of miR-146a expression, both within cells and within their secreted EVs. Further, we assessed the effects of miR-146a overexpression on bioenergetic function in primary rat glial cells and found significant reductions in oxidative phosphorylation and glycolysis. Lastly, we correlated miR-146a expression levels within various regions of the AD brain to disease staging and found significant, positive correlations. These novel results demonstrate that the modulation of miR-146a in response to neuroinflammatory stimuli may mediate the loss of mitochondrial integrity and function in cells, thereby contributing to the progression of beta-amyloid and tau pathology in the AD brain. Multiple inflammatory stimuli can upregulate miRNA-146a expression within neurons, mixed glial cells, and brain endothelial cells, which is either retained within these cells or released from them as extracellular vesicle cargo. The upregulation of miR-146a disrupts cellular bioenergetics in mixed glial cells. This mechanism may play a critical role in the neuroinflammatory response observed during Alzheimer's disease.

miR-146a Dysregulates Energy Metabolism During Neuroinflammation.

Despite decreased rates of tobacco smoking in many areas, cigarette smoking remains a major contributor to many health problems. Cigarette smoking can reduce immune system functioning while concurrently increasing inflammation. Dendritic cells in the lung exposed to cigarette smoke become stimulated and go on to activate T-cells. Extracellular vesicles (EVs) are nano-sized particles released by cells. They participate in intercellular communication by transferring functional proteins and nucleic acids to recipient cells and have been implicated in immune responses. For example, they can display MHC-peptide complexes to activate T-cells. In the current study, we sought to understand the role of cigarette smoke extract (CSE) on dendritic cell-derived EVs and their capacity to activate and differentiate T-cells. Primary human dendritic cells (iDCs) were exposed to CSE and EVs were separated and characterized. We exposed autologous primary CD4 + T-cells to iDC-EVs and observed T helper cell populations skewing towards Th1 and Th17 phenotypes. As HIV + individuals are disproportionately likely to be current smokers, we also examined the effects of iDC-EVs on acutely infected T-cells as well as on a cell model of HIV latency (ACH-2). We found that in most cases, iDC-CSE EVs tended to reduce p24 release from the acutely infected primary T-cells, albeit with great variability. We did not observe large effects of iDC-EVs or direct CSE exposure on p24 release from the ACH-2 cell line. Together, these data suggest that iDC-CSE EVs have the capacity to modulate the immune responses, in part by pushing T-cells towards Th1 and Th17 phenotypes.

Article	Year
miR-146a Dysregulates Energy Metabolism During Neuroinflammation. Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology\| PubMed ID: 34028667	2021
Cigarette smoke-induced extracellular vesicles from dendritic cells alter T-cell activation and HIV replication. Toxicology letters\| PubMed ID: 35181468	2022

Ashley E. Russell

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