Proteomic Profiling of Extracellular Vesicles Isolated From Cerebrospinal Fluid of Former National Football League Players at Risk for Chronic Traumatic Encephalopathy

Abstract

Chronic Traumatic Encephalopathy (CTE) is a tauopathy that affects individuals with a history of repetitive mild traumatic brain injury, such as American football players. Initial neuropathologic changes in CTE include perivascular deposition of phosphorylated microtubule-associated protein tau (p-tau) neurofibrillary tangles and other aggregates in neurons, astrocytes and cell processes in an irregular pattern often at the depths of the cortical sulci. In later stages, the p-tau depositions become widespread and is associated with neurodegeneration. Extracellular vesicles (EVs) are known to carry neuropathogenic molecules, most notably p-tau. We therefore examined the protein composition of EVs isolated from the cerebrospinal fluid (CSF) of former National Football League (NFL) players with cognitive and neuropsychiatric dysfunction, and an age-matched control group (CTRL) with no history of contact sports or traumatic brain injury. EVs were isolated from the CSF samples using an affinity purification kit. Total tau (t-tau) and tau phosphorylated on threonine181 (p-tau181) in CSF-derived EVs from former NFL players and CTRL participants were measured by ultrasensitive immunoassay. The t-tau and p-tau181 levels of CSF-derived EV were positively correlated with the t-tau and p-tau181 levels of total CSF in former NFL players, respectively, but not in the CTRL group. 429 unique proteins were identified from CSF-derived EVs and quantified by TMT-10 plex method. The identified protein molecules were significantly enriched for the extracellular exosome molecules, Alzheimer’s disease pathway and Age/Telomere Length ontology as determined by DAVID Gene Ontology analysis. Ingenuity pathway analysis of the differentially expressed EV proteins revealed enrichment of canonical liver/retinoid X receptor activation pathway. Upstream effect analysis predicted MAPT (tau) as an upstream regulator in former NFL players. These data will be useful for understanding the EV-mediated disease spread and development of novel EV biomarkers for CTE and related disorders.

Dysregulation of Exosome Cargo by Mutant Tau Expressed in Human-induced Pluripotent Stem Cell (iPSC) Neurons Revealed by Proteomics Analyses

“…Exosomes are secreted from neurons and many cell types, representing extracellular vesicles (50 -150 nm diameter) of endosomal origin (20 -23) which function in the removal of cellular components and transcellular shuttling of exosome cargo consisting of proteins, RNAs, lipids, and metabolites (24). Tau is present in exosomes from cerebrospinal fluid (CSF) of AD patients (25) and CTE risk cases (26). Studies of Tau in neuronally-derived exosomes isolated from plasma of AD patients indicate that levels of phosphorylated Tau (p-Tau) predict conversion of MCI (mild cognitive impairment) to AD dementia (17).…”

Extracellular Vesicles as an Efficient and Versatile System for Drug Delivery

“…Red blood cell extracellular vesicles (RBCEVs) display surface glycophorin A, a well-known RBC marker while mesenchymal stem cell (MSC)-derived EVs exhibit surface CD73, CD90, and CD105 expression pattern [ 98 , 99 , 100 ]. Besides tracing the EV origin, researchers are also exploring the possibility of using cell-type-specific proteins on EVs to discriminate disease subtypes and to predict disease prognosis [ 101 , 102 ]. EV online databases such as Vesiclepedia [ 103 ] and ExoCarta [ 104 ] contain over thousands of EV protein entries, allowing for a conglomerate of EV protein knowledge.…”

Clinical Presentation of Chronic Traumatic Encephalopathy

“…Studies of potential fluid biomarkers for the detection of CTE have included both CSF and plasma measures of neurodegeneration (e.g., total tau, 73-75 neurofilament light 75 ) and microglial activation (e.g., sTREM2 74 ), as well as exosomal measures of tau 76,77 and proteomic profiling of CSF exosomes. 78 Each of these approaches has yielded promising, though preliminary, results in need of replication and refinement.…”