Proteomic analysis of cerebrospinal fluid extracellular vesicles reveals synaptic injury, inflammation, and stress response markers in HIV patients with cognitive impairment

Abstract

BackgroundExtracellular vesicles (EVs) are nano-sized particles present in most body fluids including cerebrospinal fluid (CSF). Little is known about CSF EV proteins in HIV+ individuals. Here, we characterize the CSF EV proteome in HIV+ subjects and its relationship to neuroinflammation, stress responses, and HIV-associated neurocognitive disorders (HAND).MethodsCSF EVs isolated from 20 HIV+ subjects with (n = 10) or without (n = 10) cognitive impairment were characterized by electron microscopy, nanoparticle tracking analysis, immunoblotting, and untargeted LC/MS/MS mass spectrometry. Functional annotation was performed by gene ontology (GO) mapping and expression annotation using Biobase Transfac and PANTHER software. Cultured astrocytic U87 cells were treated with hydrogen peroxide for 4 h to induce oxidative stress and EVs isolated by ultracentrifugation. Selected markers of astrocytes (GFAP, GLUL), inflammation (CRP), and stress responses (PRDX2, PARK7, HSP70) were evaluated in EVs released by U87 cells following induction of oxidative stress and in CSF EVs from HIV+ patients by immunoblotting.ResultsMass spectrometry identified 2727 and 1626 proteins in EV fractions and EV-depleted CSF samples, respectively. CSF EV fractions were enriched with exosomal markers including Alix, syntenin, tetraspanins, and heat-shock proteins and a subset of neuronal, astrocyte, oligodendrocyte, and choroid plexus markers, in comparison to EV-depleted CSF. Proteins related to synapses, immune/inflammatory responses, stress responses, metabolic processes, mitochondrial functions, and blood-brain barrier were also identified in CSF EV fractions by GO mapping. HAND subjects had higher abundance of CSF EVs and proteins mapping to GO terms for synapses, glial cells, inflammation, and stress responses compared to those without HAND. GFAP, GLUL, CRP, PRDX2, PARK7, and HSP70 were confirmed by immunoblotting of CSF EVs from subjects with HAND and were also detected in EVs released by U87 cells under oxidative stress.ConclusionsThese findings suggest that CSF EVs derived from neurons, glial cells, and choroid plexus carry synaptic, immune/inflammation-related, and stress response proteins in HIV+ individuals with cognitive impairment, representing a valuable source for biomarker discovery.

Electrostatic Surface Properties of Blood and Semen Extracellular Vesicles: Implications of Sialylation and HIV-Induced Changes on EV Internalization

“…To our knowledge, this is the first study to report on HIV-induced alteration in body fluid EV surface charge. Our observation that HIV infection increased the negative surface charge on BEVs and SEVs ( Figure 3 c) is novel, and in line with the notion that HIV reprograms the epigenome and proteome of host cells [ 86 , 87 , 88 , 89 , 90 , 91 ] and EVs [ 51 , 92 ]. The reason for the differences in surface charge between HIV+ and HIV- BEVs and SEVs is unknown.…”

Blood Biomarkers for Detection of Brain Injury in COVID-19 Patients

“…Observational cohort study 178 elevations of BBs in CSF of tuberculous meningitis patients (GFAP, S100 calcium-binding protein [S100B], and neuronspecific enolase [NSE]), [172][173][174][175] HIV patients (NfL, GFAP, and S100B), [176][177][178][179][180] and cerebral malaria (S100B, NSE, tau proteins, and inflammatory protein markers). [181][182][183][184][185][186] Protein biomarkers of CNS damage have also been detected after a variety of acute injury modalities.…”

Characterization of brain‐derived extracellular vesicles reveals changes in cellular origin after stroke and enrichment of the prion protein with a potential role in cellular uptake

“…Since it could be that a given marker protein is more strongly sorted into sEVs than others (and thus not directly indicating the relative contribution of this cell population to the total pool of sEVs), we decided to assess two (exclusively membrane-bound) marker proteins per cell type in order to reduce the risk of misinterpretation. The G-protein coupled P2Y receptor (P2Y12) and the Transmembrane protein 119 (TMEM119) were chosen as microglial markers [56,57]; PLP (proteolipid protein, a major component of the myelin sheath) and 2′-3′-Cyclicnucleotide 3′phosphodiesterase (CNP) were used as oligodendrocyte markers [58,59]; synapsin1 and the Synaptosomal Nerve-Associated Protein 25 (SNAP25), present at the membrane of synaptic vesicles and at the pre/postsynaptic membrane respectively, were assessed as neuronal markers [60][61][62][63][64][65], and the Excitatory Amino Acid Transporters 1 and 2 (EAAT-1 and EAAT-2) were used as astrocytic markers [66]. Quantifications are shown in Figure 3B.…”