The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points.

Neuronal damage is a hallmark feature of HIV-associated neurological disorders (HANDs). Opiate drug abuse accelerates the incidence and progression of HAND; however, the mechanisms underlying the potentiation of neuropathogenesis by these drugs remain elusive. Opiates such as morphine have been shown to enhance HIV transactivation protein Tat-mediated toxicity in both human neurons and neuroblastoma cells. In the present study, we demonstrate reduced expression of the tropic factor platelet-derived growth factor (PDGF)-B with a concomitant increase in miR-29b in the basal ganglia region of the brains of morphine-dependent simian immunodeficiency virus (SIV)-infected macaques compared with the SIV-infected controls. In vitro relevance of these findings was corroborated in cultures of astrocytes exposed to morphine and HIV Tat that led to increased release of miR-29b in exosomes. Subsequent treatment of neuronal SH-SY5Y cell line with exosomes from treated astrocytes resulted in decreased expression of PDGF-B, with a concomitant decrease in viability of neurons. Furthermore, it was shown that PDGF-B was a target for miR-29b as evidenced by the fact that binding of miR-29 to the 3′-untranslated region of PDGF-B mRNA resulted in its translational repression in SH-SY5Y cells. Understanding the regulation of PDGF-B expression may provide insights into the development of potential therapeutic targets for neuronal loss in HIV-1-infected opiate abusers.

Astrocytes are known to be critical regulators of neuronal function. However, relatively few mediators of astrocyte to neuron communication have been identified. Recent advancements in the biology of extracellular vesicles have begun to implicate astrocyte derived extracellular vesicles (ADEV) as mediators of astrocyte to neuron communication, suggesting that alterations in the release and/or composition of ADEVs could influence gliotransmission. TNFα and IL-1β are key mediators of glial activation and neuronal damage, but the effects of these cytokines on the release or molecular composition of ADEVs is unknown. We found that ADEVs released in response to IL-1β (ADEV-IL-1β) and TNFα (ADEV-TNFα) were enriched with miRNAs that target proteins involved in neurotrophin signaling. We confirmed that miR-125a-5p and miR-16-5p (both enriched in ADEV-IL-1β and ADEV-TNFα) targeted NTKR3 and its downstream effector Bcl2. Downregulation of these targets in neurons was associated with reductions in dendritic growth, dendritic complexity, reduced spike rates, and burst activity. Molecular interference of miR-125a-5p and miR-16-5p prevented ADEV-IL-1β from reducing dendritic complexity, spike, and burst rates. These findings suggest that astrocytes respond to inflammatory challenge by modifying the miRNA cargo of ADEVs to diminish the activity of target neurons by regulating the translational expression of proteins controlling programs essential for synaptic stability and neuronal excitability.

Mounting evidence suggests that microRNA (miR) dysregulation contributes to neurodegenerative disorders including Parkinson’s disease (PD). MiR-34b and miR-34c have been previously shown to be down-regulated in the brains of patients with PD. Here, we demonstrate that miR-34b and miR-34c repress the expression of α-synuclein (α-SYN), a key protein in PD pathogenesis. Inhibition of miR-34b and miR-34c expression in human dopaminergic SH-SY5Y cells increased α-SYN levels and stimulated aggregate formation. Additionally, a single nucleotide polymorphism (SNP) in the 3’-UTR of α-SYN was found to lower the miR-34b-mediated repression of the protein. Our results suggest that down-regulation of miR-34b and miR-34c in the brain, as well as an SNP in the 3’-UTR of α-SYN can increase α-SYN expression, possibly contributing to PD pathogenesis.

MicroRNAs (miRNAs) have important roles in regulating a plethora of physiological and pathophysiogical processes including neurodegeneration. In both human immunodeficiency virus (HIV)-associated dementia in humans and its monkey model simian immunodeficiency virus encephalitis (SIVE), we find miR-21, a miRNA largely known for its link to oncogenesis, to be significantly upregulated in the brain. In situ hybridization of the diseased brain sections revealed induction of miR-21 in neurons. miR-21 can be induced in neurons by prolonged N-methyl--aspartic acid receptor stimulation, an excitotoxic process active in HIV and other neurodegenerative diseases. Introduction of miR-21 into human neurons leads to pathological functional defects. Furthermore, we show that miR-21 specifically targets the mRNA of myocyte enhancer factor 2C (MEF2C), a transcription factor crucial for neuronal function, and reduces its expression. MEF2C is dramatically downregulated in neurons of HIV-associated dementia patients, as well as monkeys with SIVE. Together, this study elucidates a novel role for miR-21 in the brain, not only as a potential signature of neurological disease, but also as a crucial effector of HIV-induced neuronal dysfunction and neurodegeneration.

Nuclear factor E2-related factor 2 (Nrf2) is a key transcription factor that regulates the expression of a number of antioxidant and detoxifying genes that provide cellular protection against various stressors including reactive oxygen species (ROS). Nrf2 activity is tightly regulated by a cytoplasmic inhibitory protein called Kelch-like ECH-associated protein 1 (Keap1). The mechanism that controls Keap1 expression, however, remains poorly understood. In the present study, we demonstrate that microRNA-7 (miR-7), which is highly expressed in the brain, represses Keap1 expression by targeting the 3′-untranslated region (UTR) of its mRNA in human neuroblastoma cells, SH-SY5Y. Subsequently, this event results in an increased Nrf2 activity, as evidenced by an increase in the expression of its transcriptional targets, heme oxygenase 1 (HO-1) and glutamate-cysteine ligase modifier subunit (GCLM), and an enhanced nuclear localization of Nrf2. In addition, miR-7 decreases the intracellular hydroperoxides level and increases the level of reduced form of glutathione, indicative of oxidative stress relief. We also demonstrate that targeted repression of Keap1 and activation of Nrf2 pathway, in part, underlies the protective effects of miR-7 against 1-methyl-4-phenylpyridinium (MPP+)-induced toxicity in SH-SY5Y and differentiated human neural progenitor cells, ReNcell VM. These findings point to a new mechanism by which miR-7 exerts cytoprotective effects by regulating the Nrf2 pathway.

Parkinson's disease (PD) is characterized by the progressive loss of dopaminergic neurons in the substantia nigra. Mitochondrial complex I impairment in PD is modeled in vitro by the susceptibility of dopaminergic neurons to the complex I inhibitor 1-methyl-4-phenylpyridinium (MPPϩ). In the present study, we demonstrate that microRNA-7 (miR-7), which is expressed in tyrosine hydroxylasepositive nigral neurons in mice and humans, protects cells from MPPϩ-induced toxicity in dopaminergic SH-SY5Y cells, differentiated human neural progenitor ReNcell VM cells, and primary mouse neurons. RelA, a component of nuclear factor-B (NF-B), was identified to be downregulated by miR-7 using quantitative proteomic analysis. Through a series of validation experiments, it was confirmed that RelA mRNA is a target of miR-7 and is required for cell death following MPPϩ exposure. Further, RelA mediates MPPϩ-induced suppression of NF-B activity, which is essential for MPPϩ-induced cell death. Accordingly, the protective effect of miR-7 is exerted through relieving NF-B suppression by reducing RelA expression. These findings provide a novel mechanism by which NF-B suppression, rather than activation, underlies the cell death mechanism following MPPϩ toxicity, have implications for the pathogenesis of PD, and suggest miR-7 as a therapeutic target for this disease.

Extracellular vesicles have now emerged as key players in cell‐to‐cell communication. This is particularly important in the central nervous system, where glia–neuron cross‐talk helps maintain normal neuronal function. Astrocyte‐derived extracellular vesicles (ADEVs) secreted constitutively promote neurite outgrowth and neuronal survival. However, extracellular stimuli can alter the cargo and downstream functions of ADEVs. For example, ADEVs secreted in response to inflammation contain cargo microRNAs and proteins that reduce neurite outgrowth, neuronal firing, and promote neuronal apoptosis. We performed a comprehensive quantitative proteomic analysis to enumerate the proteomic cargo of ADEVs secreted in response to multiple stimuli. Rat primary astrocytes were stimulated with a trophic stimulus (adenosine triphosphate, ATP), an inflammatory stimulus (IL‐1β) or an anti‐inflammatory stimulus (IL10) and extracellular vesicles secreted within a 2 hr time frame were collected using sequential ultracentrifugation method. ADEVs secreted constitutively without exposure to any stimulus were used a control. A tandem mass tag‐based proteomic platform was used to identify and quantify proteins in the ADEVs. Ingenuity pathway analysis was performed to predict the downstream signaling events regulated by ADEVs. We found that in response to ATP or IL10, ADEVs contain a set of proteins that are involved in increasing neurite outgrowth, dendritic branching, regulation of synaptic transmission, and promoting neuronal survival. In contrast, ADEVs secreted in response to IL‐1β contain proteins that regulate peripheral immune response and immune cell trafficking to the central nervous system.