Vesicular Transport of Encapsulated microRNA between Glial and Neuronal Cells

Exosomes (EXs) and extracellular microvesicles (EMVs) represent a diverse assortment of plasma membrane-derived nanovesicles, 30–1000 nm in diameter, released by all cell lineages of the central nervous system (CNS). They are examples of a very active and dynamic form of extracellular communication and the conveyance of biological information transfer essential to maintain homeostatic neurological functions and contain complex molecular cargoes representative of the cytoplasm of their cells of origin. These molecular cargoes include various mixtures of proteins, lipids, proteolipids, cytokines, chemokines, carbohydrates, microRNAs (miRNA) and messenger RNAs (mRNA) and other components, including end-stage neurotoxic and pathogenic metabolic products, such as amyloid beta (Aβ) peptides. Brain microglia, for example, respond to both acute CNS injuries and degenerative diseases with complex reactions via the induction of a pro-inflammatory phenotype, and secrete EXs and EMVs enriched in selective pathogenic microRNAs (miRNAs) such as miRNA-34a, miRNA-125b, miRNA-146a, miRNA-155, and others that are known to promote neuro-inflammation, induce complement activation, disrupt innate–immune signaling and deregulate the expression of neuron-specific phosphoproteins involved in neurotropism and synaptic signaling. This communication will review our current understanding of the trafficking of miRNA-containing EXs and EMVs from astrocytes and “activated pro-inflammatory” microglia to target neurons in neurodegenerative diseases with an emphasis on Alzheimer’s disease wherever possible.


Microvesicle Signaling in Neurodegeneration
Overview

Exosomes (EX) and Extracellular Microvesicles (EMV)
The exosome (EX) and extracellular microvesicle (EMV), sometimes referred to collectively as extracellular microparticles (MP) or extracellular organelles, are lipid-bilayer membrane encapsulated, nanosized particles 30-1000 nm in diameter that play essential roles: (i) in intercellular communication and tissue homeostasis by transporting diverse classes of biologically active molecules between cells of glial and neuronal origins (and probably vice versa; glial cells include microglial cells, astroglia and astrocytes); (ii) in maintaining the crosstalk between neurons and glia and neural cells with the peripheral nervous system (PNS) and systemic circulation; (iii) in synaptic plasticity, neuroprotection, neuroregeneration, maintenance of the neuronal-glial interface, and the dissemination proteus and slime molds such as Dictyostelium discoideum, nematodes such as Caenorhabditis, and up the evolutionary scale to a wide range of mammals including humans. The biogenesis, secretion and the release of EXs, MPs and/or EMVs into the extracellular space or external environment for the purpose of mediating intercellular communication or transmitting DNA-or RNA-encoded genetic information between different cell types and the environment is therefore a very ancient and conserved evolutionary process (Columbo et al., 2014 [13]; Arbo et al., 2020 [6]). From what is currently known, the EXs, MPs and/or EMVs of all prokaryotes and eukaryotes examined so far all carry small non-coding RNAs (sncRNAs), microRNA-like and/or microRNAs, as a fundamental system for the extracellular exchange and/or transmission of genetic information and there is the intriguing potential here for interspecies signaling and communication (Pogue et al. 2014 [33]; Cong et al., 2018 [34]; Avsar et al., 2020 [35]). ; both types of vesicular transport systems have been observed to operate in the brain between astroglial cells and neurons; (A) exosomes-when mature intracellular endosomes (also known as multi-vesicular bodies) containing intraluminal vesicles (ILVs; black outlined green spheres) fuse with the plasma membrane and empty their plasma membrane-encapsulated cargo, ILVs are released and, from being extracellular, they become exosomes (EX); these 30-100 nm diameter spheres contain various mixtures of proteins, lipids, proteolipids, cytokines, chemokines, microRNAs (miRNA), messenger RNAs (mRNA) and end-stage neurotoxic metabolic products, including 42 amino acid amyloid-beta (Aβ42) peptides, tau proteins and/or the lipid raft associated flotillin ( Interestingly, EX, MP and EMV releases are common features of many diverse cell types in many different kinds of organisms, from prokaryotes to eukaryotes, and their formation and release have been widely documented in the gastrointestinal (GI) tract bacteria such as Escherichia coli and Bacteroides fragilis, species of the family Brassicaceae such as Arabidopsis thaliana, Protists, protozoa such as Amoeba proteus and slime molds such as Dictyostelium discoideum, nematodes such as both types of vesicular transport systems have been observed to operate in the brain between astroglial cells and neurons; (A) exosomes-when mature intracellular endosomes (also known as multi-vesicular bodies) containing intraluminal vesicles (ILVs; black outlined green spheres) fuse with the plasma membrane and empty their plasma membrane-encapsulated cargo, ILVs are released and, from being extracellular, they become exosomes (EX); these 30-100 nm diameter spheres contain various mixtures of proteins, lipids, proteolipids, cytokines, chemokines, microRNAs (miRNA), messenger RNAs (mRNA) and end-stage neurotoxic metabolic products, including 42 amino acid amyloid-beta (Aβ42) peptides, tau proteins and/or the lipid raft associated flotillin (Angelopoulou et al. 2020 [29]; Hornung et al., 2020 [30]); EXs appear to play a central role in the spread of Aβ42 pathology and amyloidogenesis EXs and EMVs: (i) are released under normal physiological conditions, but are also discharged from parent cells upon cellular activation, hypoxia and/or hyperoxia, senescence, apoptosis and disease via a paracrine-and endocrine-type type action to their target cells; (ii) represent one of the major biological mechanisms for genetic exchange, immune signaling and the spread of inflammation and disease between cells of the host; (iii) EX and EMV trafficking in the mammalian CNS is a particularly robust, active and dynamic process (Valadi et

Extracellular Trafficking of EX and EMV Cargo
The biogenesis of EXs and EMVs, and the triggers for their release from parent cells, are complex biological processes that eventually require the navigation of these vesicles through the extracellular matrix to their extracellular targets which may be near or distant (Barnes and Somerville 2020 [7]; Birgisdottir and Johansen 2020 [41]; BriteS 2020 [42]; Jadli et al., 2020 [43]). Protein interaction maps have indicated that EX and EMV biolipid boundary membranes have a tendency to interact with discrete extracellular "node" proteins involving surface ligands and delivery receptors, indicating an "extracellular directional strategy" for vesicular sorting and translocation over both short and long distances (Choi et al., 2015 [44]; Leidal and Debnath 2020 [45]). Many of these directional pathways and sorting mechanisms remain incompletely understood. EX and EMV targets may be either locally or distally located within the CNS or, after the passage of vesicles from the CNS into the systemic circulation, vesicular cargoes may target extra-neural cell receptors throughout the CNS (Barbagallo et al., 2020 [46]; Serpente et al., 2020 [11]).
The dynamics and observed variability of EXs and EMVs in vesicle size, morphology and cargo further indicates: (i) that these vesicular organelles consist of a unique repertoire of cytoplasmic components representing cellular, molecular and genetic information that is a direct reflection of the unique biological condition of the parent cell's cytoplasm at the time of vesicular release; (ii) that these microparticles play important roles as enveloped proteolipids, a nucleic acid-enriched "information packet" in a complex extracellular communication network; (iii) that EXs and EMVs may reprogram recipient, adjacent cells and/or distant tissues as CNS-resident cells involved in immune-surveillance and the maintenance of normal cellular homeostasis, while also contributing to neuropathology during disease; (iv) that the molecular content and rates of production and secretion of EXs and EMVs vary greatly depending on the cell-type and physiological state of the cells of vesicular origin (van Niel et al., 2018 [47]; Mathieu et al., 2019 [15]; Brites 2020 [42]; Leidal and Debnath 2020 [45]). Attesting to the importance of EXs and EMVs in neurocellular and CNS functions, it is remarkable that the genetic and pharmacological inhibition or content modification of EX or EMV secretion from astrocytes has been found to play a role in the induction of amyloidogenesis, inflammation status and disease progression in several transgenic models of murine neurodegeneration, including the aging, amyloid-overexpressing 5xFAD mouse model (Dinkins et al., 2016 [48]; Barnes and Somerville 2020 [7]; Leidal and Debnath 2020 [45]).
Some miRNAs, such as the neurologically relevant miRNA-378, are very highly conserved, and their core ribonucleotide sequence has remained virtually unchanged in plants (Atropa belladonna) and higher animals (Homo sapiens) over many billions of years of evolution (Arabidopsis thaliana-Homo sapiens divergence~1. 5  The discovery of the important role of miRNAs in the regulation of the transcriptome of a cell was made about~15 years ago and was shortly followed by the first reports of altered miRNA abundance, speciation and complexity in the limbic system of Alzheimer's disease (AD) brains (Lukiw 2007 [60]) This included a significant up-regulation of what are now known as "pro-inflammatory miRNAs", including miRNA-34a, miRNA-125b, miRNA-146a, miRNA-155 and others in the parenchyma of the temporal lobe neocortex (Lukiw 2007 [28]). The first reports of EXs and EMVs being loaded with ribonucleic acid cargoes, such as specific miRNAs and mRNAs, first came about 12 years ago: (i) from the microarray analysis of mast cells involved in innate and adaptive immunity, autoimmunity, and inflammation (Valadi et al., 2007 [26]); (ii) from studies of specific miRNAs, mRNAs and angiogenic proteins in glioblastoma and neuroblastoma tumor cells that have released EXs and EMVs (Skog et al., 2008 [25]; Columbo et al., 2014 [13]; Briand et al., 2020 [8]). These extruded vesicles were subsequently observed to be taken up by normal host brain microvascular endothelial cells, thereby inducing carcinogenic type phenotypic change in these brain cells, stimulating tumor invasion, proliferation and cancer spread mediated by endothelial cells of the neurovasculature (Hunter et al., 2008 [36]; Skog et al., 2008 [25]). Very recently, evidence has been provided showing that EXs and EMVs derived from miRNA-containing natural killer (NK) cells contribute to immunological surveillance and their provision of specific miRNAs may be the first-line of defense in the regulation of tumor cell growth, cytotoxicity and metastasis diffusion in the CNS (Briand et al., 2020 [8]; Federici et al., 2020 [62]). As discussed more fully below, it has recently been shown that brain EXs and EMVs released from diseased astrocytes and "activated microglia" carry specific miRNA-enriched cargoes, enriched in miRNA-34a and miRNA-125b for example, that contribute to neuropathological spreading and the exacerbation of neuropathological processes in AD, ALS, Parkinson's disease (PD), Huntington's disease (HD), stroke and other neuro-inflammatory degenerative conditions. This indicates a definitive role for EX and EMV miRNA cargoes in neurological disease processes with an inflammatory component which may have considerable diagnostic, prognostic and/or therapeutic value (Prada et [64]). Because brain cells can release EXs and EMVs which can pass from the brain and CNS into the blood, brain-derived vesicles isolated from the systemic circulation may be of use to monitor diseases operating in the CNS, thus improving clinical diagnoses and prognoses (Barbagallo et al., 2020 [46]). This may also be of use in analyzing the efficacy of pharmaceuticals and/or therapeutic interventions being used to intervene in the AD process via the analysis of EXs and EMVs in blood plasma (Cha et al., 2019 [65]; Barbagallo et al., 2020 [46]).

EMV and miRNA Cargoes in the Spreading of Inflammatory Neurodegeneration
Accumulating evidence continues to implicate secreted miRNAs, including EX and EMV-encapsulated miRNAs, in the pathogenic spreading of progressive, age-related and incapacitating neurodegenerative diseases with an uncontrolled or deregulated inflammatory component and synaptic deficits.  [70]). From extensive research reports, it seems most likely that, in inflammatory neurodegenerative disease, the up-regulation of single miRNAs is much less important than increases in small families of pathological miRNAs in driving disease progression (Zhao et al., 2015 [61]; Lukiw 2020 [70]). While the alterations in the abundance of select miRNAs may be disease-specific, others such as miRNA-125b, miRNA-146a and miRNA-155 appear to be up-regulated during many of these neurological disorders and play highly integrated pathological roles. For example, an increase of miRNA-146a and miRNA-155 is observed in AD and AMD. Both miRNA-146a and miRNA-155 target the 3 untranslated region (3 -UTR) of the complement factor H (CFH) mRNA in overlapping binding sites leading to a highly effective down-regulation in the expression of CFH and runaway loss-of-complement control, markedly increased pro-inflammatory signaling thereby contributing mechanistically to both AD and AMD inflammatory neuropathology (Hill et al., 2015 [30]; Fan et al., 2020 [68]).
Importantly, the modification of miRNA expression to more homeostatic levels via anti-miRNA or anti-NF-kB strategies may be a useful therapeutic strategy to successfully address multiple aspects of neuropathological inflammation (Hammond 2015 [28]; Zhao et al., 2016 [66]; Ghaffari et al., 2020 [27]). CNS-derived EXs and EMVs that contain NF-kB-sensitive, pro-inflammatory miRNAs and other molecular biomarkers from the cells of their parental origin are known to cross the blood-brain barrier into the systemic circulation and may be reflective of the biochemical status for various neurodegenerative diseases. There are practical challenges associated with the methodology of the extraction and characterization of CNS-derived blood, CSF and tissue EXs and EMVs, and subsequent analysis of their miRNAs and other intra-vesicular cargoes, however the methodologies for EX and EMV isolation and categorization are constantly improving (Hornung et al., 2020 [71]; Serpente et al., 2020 [11]).
As previously discussed, EXs or EMVs and their miRNA cargoes may be shed into the extracellular environment under physiologically homeostatic or pathological conditions, either constitutively, or upon activation via acute injury (Kandiyil et al., 2019 [72]), hypoxia, hypoxia-induced oxidative stress and the generation of reactive oxygen species (ROS) by multiple mechanisms (Deng et al., 2018 [34]), by senescence or "lingering death" (Urbanelli et al., 2016 [20]), by pro-inflammatory mediators (including inflammatory cytokines, chemokines and Aβ peptides), by factors in the systemic circulation such as thrombin and thrombotic factors (Hunter et al., 2019 [73]), by components of the purinergic pathways (Ludwig et al., 2020 [74]) and microbial or viral virulence factors, including bacterial exotoxins and lipopolysaccharide (LPS; Mao et al., 2015 [75]; Lukiw et al., 2018 [76]; Alexandrov et al., 2019 [58]). As observed in cancer, inflammatory bowel diseases and neurodegenerative diseases, including AD and ALS, exosomes can carry adenosine receptors and other components of purinergic pathways, and the production and release of some EXs or EMVs may be induced by the stimulation of purinergic receptors by purine receptor agonists and reduced by purine receptor antagonists (Hosaka et al., 2019 [54]; Ludwig et al., 2020 [74]).

Unanswered Questions
Several important unanswered questions remain in our understanding of the generation and characterization of intraluminal vesicles (ILV), microparticles (MP), exosomes (EX) and extracellular microvesicles (EMV), the nature of their vesicular cargoes and miRNA composition. These include: (i) the signals and pathways essential for stimulation and the origin of their formation, as well as the mechanism of their release from many different cell types in the CNS and their proficiency for modulating functions of target cells; (ii) the molecular-genetic injury and/or environmental factors which stimulate their release for this evolutionarily-conserved type of information communication system amongst astrocytes, microglia and neurons; (iii) their increased production and release during the initiation and spread of progressive age-related inflammatory neurodegeneration; (iv) the actual molecular contents, stoichiometry and packaging of the contents in the vesicles themselves; (v) the magnitude and signaling impact of their plasma membrane-packaged vesicular cargo; (vi) the regulation of their trafficking and targeting to neuronal cells via plasma membrane-mediated cell-surface reception mechanisms; (vii) the lipidomic, proteomic and transcriptomic profiles of these vesicles and what miRNA and/or mRNA encoded information these vesicles may be carrying; (viii) whether or not EXs and/or EMVs can transfer their miRNA-enriched intraluminal cargoes to other cell types and/or to other species; (ix) the role of circular RNA (circRNA) which have been shown in some cases to act as natural "anti-miRNA sponges" of specific miRNA activities ( [11]). A single microvesicle, for example, may carry a cargo consisting of a complex cocktail of multiple proteins, proteolipids, cytokines, chemokines, carbohydrates, miRNAs, mRNAs, circRNAs and/or other nucleic acid signals as well as neurotrophic and amyloidogenic factors together representing novel, unique and discrete "information packets" for the structural and functional support of target cells, and acting as unique mediators of intercellular information transfer.

Concluding Remarks
The neurobiology of EX and EMV genesis, release, translocation and uptake by target cells, and their containment of select miRNA populations enriched in the CNS, indicate that they are significant components of a highly dynamic system of intercellular communication via extracellular translocation and targeting in brain cell health, aging and disease. Multiple independent studies indicate that while vesicle-mediated intercellular signaling is important in the homeostatic maintenance of brain cell functions, they have a substantial role in the proliferation of injury, cancer and inflammatory neurodegeneration signaling, as is observed in the AD brain. Many of the details of the mechanisms by which EXs and EMVs and their miRNA cargoes are generated and released by the activation of astrocytes and microglia and their trafficking to target brain cells, primarily neurons, remain to be further clarified. A greater understanding of the mechanisms underlying EX and EMV biogenesis, cargo selection and loading, vesicle release, translocation exterior to the cells of origin and targeting to adjacent or distant neural cells remains critical for unlocking the immense neurobiological and therapeutic potential for these ubiquitous organelles. Not only could an increased understanding of EX and EMV systems and their containment of miRNAs in the brain be useful in the treatment of CNS injuries and progressive age-related inflammatory neurodegeneration, but may also prove useful as delivery vehicles for therapeutic miRNAs, anti-miRNAs and both bioactive and neuroactive pharmaceuticals.