Isolation of exosomes from brain can serve as a confirmation that processes observed in the in vitro model also occur in vivo in the brain. For example, one study identified a novel mechanism that regulates the activity of the membrane-bound receptor for platelet-derived growth factor A (PDGFRα), crucial for controlling the production of oligodendrocytes for myelination (Pituch et al., 2015). This study tested whether galactosylceramides (sulfatides) contribute to the regulation of oligodendrogenesis by modulating PDGFRα function. It was found that increased sulfatides levels in multipotential neural precursors lead to a reduced production of oligodendrocytes progenitor cells and oligodendrocytes. The activity of PDGFRα depends on its localization to detergent-resistant membrane domains in the plasma membrane and interaction with integrin complexes and therefore, the diminished association of PDGFRα with these domains due to increased sulfatides levels resulted in exacerbated PDGFRα activity. This study identified a novel mechanism that regulates the secretion of PDGFRα in multipotential neural precursor cells, in glial cells, and in the brain cortex via exosomal shedding. While the in vitro data has shown that PDGFRα is secreted by multipotential neural precursors and by glial cells via exosomes, the in vivo study has shown that PDGFRα is secreted via exosomes during the peak of myelination (Pituch et al., 2015). Exosomal PDGFRα secretion may be a physiologically relevant function of exosomal secretion by which oligodendrocytes progenitor cells rapidly and locally regulate the concentration of the receptor. This can have an impact on the receptor-mediated responses to proliferation, survival and differentiation. Thus, the in vivo studies provide further evidence that exosomal regulation of PDGFRα is part of a larger regulatory program involved in myelination.

Extensive research has shown that several metabolites of the APP are toxic to neuronal cells. A central pathological feature of AD is the accumulation of Aβ in the brain, and it was suggested that Aβ has an important role in the pathogenesis of neuronal dysfunction in the disease (reviewed in Selkoe, 1991; Hardy and Selkoe, 2002; Stine et al., 2003). Recent in vitro and in vivo studies show that other APP metabolites, mainly the β-secretase derived APP-CTFs, are neurotoxic and can cause memory loss (Jiang et al., 2010; Deyts et al., 2012; Tamayev and D'Adamio, 2012; Tamayev et al., 2012). Most significantly, it was shown that APP-βCTF cause neuronal endosomal-lysosomal abnormalities and resulting memory loss (Neve et al., 1996; Oster-Granite et al., 1996; Neve and Robakis, 1998; McPhie et al., 2001; Jiang et al., 2010; Deyts et al., 2012; Flammang et al., 2012; Lauritzen et al., 2012, 2016; Oules et al., 2012; Tamayev and D'Adamio, 2012; Tamayev et al., 2012). Exosomal proteins were found to accumulate in the amyloid plaques in the brain of AD patients (Rajendran et al., 2006) and therefore it was proposed that exosomes participate in the pathogenesis of AD. Exosomes isolated from the conditioned medium of neuronal cell cultures contain as cargo the full-length APP, APP metabolites, and key enzymes that cleave both full-length APP and CTFs, and exosomes are one site where APP processing occurs (Rajendran et al., 2006; Vingtdeux et al., 2007; Escrevente et al., 2008; Sharples et al., 2008). The presence of precursors to amyloidogenic proteins and amyloidogenic proteins within exosomes suggest that exosomes can release amyloidogenic material and transmit it between cells, playing a pathogenic role in neurodegenerative diseases.

Isolation of exosomes from mouse brains had shown that murine brain exosomes contain full-length APP and the APP metabolites APP-CTFs and Aβ (Perez-Gonzalez et al., 2012) as was shown previously for exosomes isolated from the media cultured by neuronal cells in vitro (Vingtdeux et al., 2007). A novel finding was that exosomes isolated from brains of mice are enriched with APP-CTFs compared to brain homogenates (Perez-Gonzalez et al., 2012). These results imply the existence of a set of mechanisms driving an enrichment of APP-CTFs in exosomes. Additionally brain exosomes were isolated from brains of transgenic mice overexpressing human APP with the K670N/M671L Swedish double mutation (Tg2576; Hsiao et al., 1996). The findings showed that the amount of flAPP and APP-CTFs secreted out of the cell in brain exosomes is proportional to their expression levels in the brain, but that the exosomes are greatly enriched with APP-CTFs (Perez-Gonzalez et al., 2012). Immuno-EM using antibodies directed to cytoplasmic or extracellular epitopes of APP revealed that the exosomes are oriented with the cytoplasmic-side facing inward (Pisitkun et al., 2004). Therefore, cleavage of APP results in capture of APP-CTFs inside exosomes. Thus, the enrichment of APP-CTFs in exosomes isolated from brains results from endosomal APP-CTFs that were captured and carried by exosomes combined with metabolism of flAPP in the exosomes. Contrary to flAPP and APP-CTFs, only a minute fraction of total Aβ (<1%) is associated with exosomes (Rajendran et al., 2006). The orientation of APP in the membrane of exosomes would result in the secretion of exosomal-generated Aβ into the extracellular space, not into the vesicles. Data suggest that Aβ binds to exosomes that possibly act as a seed for plaque formation (reviewed in Dinkins et al., 2016a).

Isolation of exosomes from murine brain also enables direct in vivo study of the effect of exosomes release on amyloidogenesis. This was studied in a transgenic mouse expressing five familial mutations in the human APP and presenilin 1 genes (5XFAD). This model primarily produces Aβ42, with amyloid plaques forming as early as at 3 months of age (Oakley et al., 2006). Prevention of exosomes secretion by the inhibition of neutral sphingomyelinase 2 (nSMase2), a key regulatory enzyme generating ceramide from sphingomyelin, with GW4869 in the 5XFAD transgenic mouse reduced total Aβ42 levels and plaque load in the brain (Dinkins et al., 2014). In genetically nSMase2-deficient 5XFAD mice, reduced glial activation, total Aβ42 and plaque burden, and tau phosphorylation, along with an improvement in a fear-conditioned learning task were observed (Dinkins et al., 2016b). These findings are consistent with the idea that APP metabolite loaded exosomes may, under some conditions, contribute to amyloidogenesis. Additionally, the isolation of exosomes from mouse brain facilitated the identification of a difference between females and males, showing that the reduction of total Aβ42 levels and the number of plaques in the brain with a reduction in nSMase2 function was significantly greater in male than female mice (Dinkins et al., 2014), suggesting sex differences in brain exosome secretion or function. This information may provide a mechanism for the higher vulnerability of females to AD pathogenesis: sex differences have been reported in AD, with evidence that women expressing the apolipoprotein E4 allele have a higher risk of being affected by AD than men, regardless of longevity and other disease mortality factors (Payami et al., 1994; Buckwalter et al., 1996; Farrer et al., 1997; Andersen et al., 1999; Bretsky et al., 1999; Lobo et al., 2000; Mortensen and Hogh, 2001; Brookmeyer et al., 2002; Barnes et al., 2003; Baum, 2005; Beydoun et al., 2010; Altmann et al., 2014).

Another major hallmark of AD is the accumulation of pathological tau protein, spreading in well-defined stages between different regions of the brain, potentially via exosomes. Exosomes-associated tau phosphorylated at Thr-181 (AT270), an established phosphorylated tau biomarker for AD, was found in tissue culture media and in human CSF samples, suggesting that exosomes-mediated secretion of phosphorylated tau plays a role in the abnormal processing of tau in early AD (Saman et al., 2012). It was hypothesized that microglia, the primary phagocytes in the brain, facilitate tau protein propagation between neurons by phagocytosing and exocytosing tau, supported by the finding that depleting microglia dramatically suppressed the propagation of tau (Asai et al., 2015). An in vitro study had shown that microglia are more efficient at the uptake of tau as compared to neurons and astrocytes. Isolation of exosomes from the brain of a mouse model of tau propagation revealed that vesicles that contain exosomal proteins contain tau and that exosomes isolated from the brain of these mice transduce tau into primary cultured neurons (Asai et al., 2015). Moreover, microglial depletion partially reduced tau content in exosomes, and inhibiting exosomes synthesis significantly reduced tau propagation in vitro and in vivo. These data suggest that exosomes may play a prominent role in the progression of tauopathy within the brain (Asai et al., 2015).

A study that isolated exosomes from the brain of a tau transgenic mouse that expresses four-repeat tau with the P301L mutation showed that exosomes levels of tau were significantly higher in transgenic mice that have pronounced tau pathology (Polanco et al., 2016). Tau in the vesicles was differentially phosphorylated, although to a lower degree than in the brain cells from which they were derived. Several phospho-epitopes (AT8, AT100, and AT180) thought to be critical for tau pathology were undetected in brain exosomes. Despite this, exosomes derived from transgenic mice were capable of seeding tau aggregation in a threshold-dependent manner, demonstrating that tau-containing exosomes can transmit tau pathology. Moreover, the characteristics of tau in the exosomes and the high seeding threshold identified may explain why tau pathology develops very slowly in neurodegenerative diseases such as AD (Polanco et al., 2016). In another study, the seeding potential of lysates and EVs enriched for exosomes from wild-type and the tau transgenic mouse brains was compared, showing that the transgenic exosomes cause increased tau phosphorylation and soluble oligomer formation in a manner comparable to that of freely available proteins in brain lysates, a prerequisite for the formation of mature protein aggregate (Baker et al., 2016). These in vivo studies show that exosomes can propagate tau pathology, can carry tau seeds able to induce tau aggregation in recipient cells, and that such tau is phosphorylated at epitopes found in AD patients.

α-synuclein aggregation plays a central role in Parkinson's disease pathology. Exosomes released from α-synuclein overexpressing SH-SY5Y cells contain α-synuclein, and these exosomes were capable of efficiently transferring α-synuclein to normal SH-SY5Y cells (Emmanouilidou et al., 2010; Alvarez-Erviti et al., 2011). An in vitro study that examined the role of the ESCRT pathway in the propagation of α-synuclein has shown that disruption of the ESCRT transport system resulted in increased exocytosis of α-synuclein (Spencer et al., 2016). The in vitro study also showed that the ESCRT protein-charged multivesicular body protein (CHMP2B) affects the release of α-synuclein, and downregulation of CHMP2B affects the intracellular capacity of cells to clear α-synuclein, leading to greater release of α-synuclein into the media. Consistent with these findings, lower levels of CHMP2B were found in the brain of patients diagnosed with dementia with Lewy body as compared with controls, thus generating a roadblock of endocytosed α-synuclein. In order to determine whether α-synuclein is released via exosomes in vivo, exosomes were isolated from brain of patients with dementia with Lewy body, revealing that the exosomes contained α-synuclein, while exosomes isolated from control individuals did not (Spencer et al., 2016). These data suggest that neurons compensate for dysfunction of the endosomal-lysosomal pathway that cause accumulation of material such as α-synuclein in MVB by enhanced release of exosomes, potentially increasing cell-to-cell propagation of α-synuclein in neurological disorders with parkinsonism, including Parkinson's disease and dementia with Lewy body.

Similar to dementia with Lewy body, data on exosomes-associated TDP-43, a pathological hallmark of amyotrophic lateral sclerosis and frontotemporal lobar degeneration, suggest that enhanced exosomes release is beneficial to neuronal survival, in spite of potential increase in disease propagation (Iguchi et al., 2016). in vitro investigation of the role of exosomes in the secretion and propagation of TDP-43 aggregates have shown that TDP-43 is secreted by neurons but not by astrocytes or microglia in association with exosomes. Isolation of exosomes from human brains had shown that levels of exosomal TDP-43 full length and C-terminal fragment species are upregulated in human amyotrophic lateral sclerosis brains. When Neuro2a cells were exposed to exosomes from amyotrophic lateral sclerosis brain, but not from control brain, cytoplasmic redistribution of TDP-43 was observed (Iguchi et al., 2016). These data suggest that secreted exosomes contribute to propagation of TDP-43 proteinopathy. However, inhibition of exosomes secretion provoked formation of TDP-43 aggregates in Neuro2a cells and exacerbated TDP-43 neuronal aggregation. Moreover, the disease phenotypes of transgenic mice expressing human TDP-43A315T mutant was exacerbated following in vivo inhibition of exosomes secretion. Given that cytoplasmic TDP-43 aggregation occurs in amyotrophic lateral sclerosis and frontotemporal lobar degeneration, these results suggest that exosomes containing pathological TDP-43 play a role in the propagation of TDP-43 proteinopathy. Thus, a therapeutic strategy for amyotrophic lateral sclerosis based on inhibition of exosomes production based on in vivo data that suggest that exosomes secretion plays an overall beneficial role in neuronal clearance of pathological TDP-43 would be inadvisable (Iguchi et al., 2016).

Investigation of the role of exosomes in simian immunodeficiency virus (SIV)-induced brain disease was conducted by the isolation and characterization of exosomes from the brains of rhesus macaques. Small RNA sequencing revealed higher miR-21 levels in exosomes from SIV encephalitic brains compared with controls (Yelamanchili et al., 2015a,b). In situ hybridization revealed increased miR-21 expression in neurons and macrophage/microglial cells/nodules during SIV induced brain disease. in vitro culture of macrophages have shown that miR-21 is released via exosomes and is neurotoxic via activation of TLR7 when compared to exosomes derived from miR-21 knockout mice (Yelamanchili et al., 2015a,b). This study further emphasizes the value of the characterization of exosomal response in the affected brain in combination with an in vitro study to further define the mechanism of action.

The same exosomes-associated miRNA involved in SIV-induced brain disease was found to have a role in traumatic brain injury (TBI; Harrison et al., 2016). While several experiments have shown the effects of exosomes-associated miRNA on cells in culture, this study investigated how miRNA content of exosomes is altered in vivo in the brain. Exosomes were isolated from the brain of a mouse model of TBI (controlled cortical impact) and controls. Profiling of miRNA in the exosomes has shown changes in miRNA species, with miR-21 showing the largest change between conditions. Highlighting the value of isolating exosomes from the brain directly, the miRNA profiles found in this study were not identical to those seen in exosomes isolated from human CSF after TBI described in a previous study (Patz et al., 2013). It remains to be determined whether the difference between the studies is due solely to a difference between CSF and brain exosomes, or due to a difference between TBI in humans and the mouse model, critical to the interpretation of further studies. Study of the mouse brain has shown that the expression of miR-21 in the brain was primarily localized to neurons, not microglia, near the lesion site, while adjacent to these miR-21-expressing neurons were activated microglia. The concurrent elevation of miR-21 in neurons with the increase in miR-21 in exosomes suggests that miR-21 secreted from neurons in association with exosomes is a potential mechanism of cell-to-cell communication resulting in microglia activation as a protective mechanism. Thus, while a neurotoxic function of exosomes-associated miR-21 was shown in the brain of SIV-induced rhesus macaques brain disease (Yelamanchili et al., 2015a,b), a protective function is suggested, but needs to be proven, in a mouse model of TBI (Harrison et al., 2016).

In another study, well-preserved exosomal miRNAs were isolated from frozen postmortem prefrontal cortices and submitted to RNA profiling to examine whether they reflect disease-specific changes in psychiatric disorders (Banigan et al., 2013). The expression of certain exosomal miRNA in the brains of patients diagnosed with schizophrenia and bipolar disorder differed from controls, reflecting either disease-specific or common aberrations in these patients. RT-PCR validation confirmed that two miRNAs, miR-497 in schizophrenia brain samples and miR-29c in bipolar disorder brain samples, have significantly higher expression when compared to control samples (Banigan et al., 2013). These results show that brain exosomes characterization has the potential to identify the cellular mechanism of disease pathology, as well as showing the potential of exosomes-derived miRNAs to serve as biomarkers for various disorders.

The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.