Assessing Neuronal and Astrocyte Derived Exosomes From Individuals With Mild Traumatic Brain Injury for Markers of Neurodegeneration and Cytotoxic Activity

Abstract

Mild traumatic brain injury (mTBI) disproportionately affects military service members and is very difficult to diagnose. To-date, there is currently no blood-based, diagnostic biomarker for mTBI cases with persistent post concussive symptoms. To examine the potential of neuronally-derived (NDE) and astrocytic-derived (ADE) exosome cargo proteins as biomarkers of chronic mTBI in younger adults, we examined plasma exosomes from a prospective longitudinal study of combat-related risk and resilience, marine resiliency study II (MRSII). After return from a combat-deployment participants were interviewed to assess TBI exposure while on deployment. Plasma exosomes from military service members with mTBI (mean age, 21.7 years, n = 19, avg. days since injury 151), and age-matched, controls (deployed service members who did not endorse a deployment-related TBI or a pre-deployment history of TBI; mean age, 21.95 years, n = 20) were precipitated and enriched against a neuronal adhesion protein, L1-CAM, and an astrocyte marker, glutamine aspartate transporter (GLAST) using magnetic beads to immunocapture the proteins and subsequently selected by fluorescent activated cell sorting (FACS). Extracted protein cargo from NDE and ADE preparations were quantified for protein levels implicated in TBI neuropathology by standard ELISAs and on the ultra-sensitive single molecule assay (Simoa) platform. Plasma NDE and ADE levels of Aβ42 were significantly higher while plasma NDE and ADE levels of the postsynaptic protein, neurogranin (NRGN) were significantly lower in participants endorsing mTBI exposure compared to controls with no TBI history. Plasma NDE and ADE levels of Aβ40, total tau, and neurofilament light (NFL), P-T181-tau, P-S396-tau were either undetectable or not significantly different between the two groups. In an effort to understand the pathogenetic potential of NDE and ADE cargo proteins, neuron-like cultures were treated with NDE and ADE preparations from TBI and non-TBI groups. Lastly, we determined that plasma NDE but not ADE cargo proteins from mTBI samples were found to be toxic to neuron-like recipient cells in vitro. These data support the presence of markers of neurodegeneration in NDEs of mTBI and suggest that these NDEs can be used as tools to identify pathogenic mechanisms of TBI.

CNS-Derived Blood Exosomes as a Promising Source of Biomarkers: Opportunities and Challenges

“…Moreover, concentration levels of Aβ42 in astrocytic exosomes were lower in AD samples compared to the concentrations in healthy control samples, whereas pT181-tau, pS396-tau, and Aβ42 concentration in neuronal exosomes were significantly higher than in the control samples (Goetzl et al, 2016). In a recent study, Rissman's group showed that plasma-derived neuronal and astrocytic exosomes from patients with mild traumatic brain injury (mTBI) contained high levels of Aβ42 and low levels of neurogranin compared to healthy individuals with no history of TBI, suggesting that injury-associated proteins in these exosomes could be used as biomarkers for mTBI (Winston et al, 2019).…”

“…CNS-derived exosomes isolated from blood also have been shown to be a useful source of biomarkers for other neurological conditions, including stroke (Chen et al, 2016) and TBI (Winston et al, 2019), and for following brain processes that are not easily accessible, such as adult hippocampal neurogenesis (AHN) (Luarte et al, 2017). The enrichment of exosomes derived from specific brain cell types, such as neurons, astrocytes, and recently oligodendrocytes by immuno-capture likely will provide a more specific and useful source of diagnostic and progression biomarkers compared to plasma or serum themselves or total exosomes isolated from these biofluids.…”

Optimized method for extraction of exosomes from human primary muscle cells

“…The absence of medium supplementation, and the lack of necroptosis and apoptosis mean that the culture medium of differentiated human muscle cells is a non-complex sample, and is therefore well-suited to the protocol described here, as opposed to serum which includes many different types of vesicle and a relatively complex molecular milieu, thereby making it difficult to isolate exosomes by size and density alone, and requiring additional approaches such as exosome pull-down to maximise purity [54,55], but leading to the analysis of a specific circulating exosome subpopulation.…”

Optimized method for extraction of exosomes from human primary muscle cells

“…The absence of medium supplementation, and the lack of necroptosis and apoptosis mean that the culture medium of differentiated human muscle cells is a non-complex sample, and is therefore wellsuited to the protocol described here, as opposed to serum which includes many different types of vesicle and a relatively complex molecular milieu, thereby making it di cult to isolate exosomes by size and density alone, and requiring additional approaches such as exosome pull-down to maximise purity [61,62], but leading to the analysis of a speci c circulating exosome subpopulation.…”