# CENTRAL NERVOUS SYSTEM EXTRACELLULAR VESICLES

EDITED BY : Dominic Martin Walsh, Francesc Xavier Guix and Grant Thomas Corbett PUBLISHED IN : Frontiers in Neuroscience

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ISSN 1664-8714 ISBN 978-2-88963-953-3 DOI 10.3389/978-2-88963-953-3

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# CENTRAL NERVOUS SYSTEM EXTRACELLULAR VESICLES

Topic Editors:

Dominic Martin Walsh, Harvard Medical School, United States Francesc Xavier Guix, Severo Ochoa Molecular Biology Center (CSIC-UAM), Spain Grant Thomas Corbett, Harvard Medical School, United States

Citation: Walsh, D. M., Guix, F. X., Corbett, G. T., eds. (2020). Central Nervous System Extracellular Vesicles. Lausanne: Frontiers Media SA. doi: 10.3389/978-2-88963-953-3

# Table of Contents

*05 Therapeutic Potential of Extracellular Vesicles for the Treatment of Nerve Disorders*

Luisa R. Galieva, Victoria James, Yana O. Mukhamedshina and Albert A. Rizvanov

*14 Leukocyte Derived Microvesicles as Disease Progression Biomarkers in Slow Progressing Amyotrophic Lateral Sclerosis Patients*

Daisy Sproviero, Sabrina La Salvia, Federico Colombo, Susanna Zucca, Orietta Pansarasa, Luca Diamanti, Alfredo Costa, Luca Lova, Marta Giannini, Stella Gagliardi, Eliana Lauranzano, Michela Matteoli, Mauro Ceroni, Andrea Malaspina and Cristina Cereda

*23 Co-cultures of Glioma Stem Cells and Primary Neurons, Astrocytes, Microglia, and Endothelial Cells for Investigation of Intercellular Communication in the Brain*

Zhiyun Wei, Shubham Kale, Rachid El Fatimy, Rosalia Rabinovsky and Anna M. Krichevsky

*31 Neuron-Derived Exosome Proteins May Contribute to Progression From Repetitive Mild Traumatic Brain Injuries to Chronic Traumatic Encephalopathy*

Edward J. Goetzl, Aurélie Ledreux, Ann-Charlotte Granholm, Fanny M. Elahi, Laura Goetzl, Jade Hiramoto and Dimitrios Kapogiannis

Kae M. Pusic, Lisa Won, Richard P. Kraig and Aya D. Pusic

*54 Assessing Neuronal and Astrocyte Derived Exosomes From Individuals With Mild Traumatic Brain Injury for Markers of Neurodegeneration and Cytotoxic Activity*

Charisse N. Winston, Haylie K. Romero, Maya Ellisman, Sophie Nauss, David A. Julovich, Tori Conger, James R. Hall, Wendy Campana, Sid E. O'Bryant, Caroline M. Nievergelt, Dewleen G. Baker, Victoria B. Risbrough and Robert A. Rissman

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

Satoshi Muraoka, Mark P. Jedrychowski, Harutsugu Tatebe, Annina M. DeLeo, Seiko Ikezu, Takahiko Tokuda, Steven P. Gygi, Robert A. Stern and Tsuneya Ikezu

*80 Considerations and Implications in the Purification of Extracellular Vesicles – A Cautionary Tale*

Yi Xin Fiona Lee, Henrik Johansson, Matthew J. A. Wood and Samir El Andaloussi

*102 miR-212 and miR-132 are Downregulated in Neurally Derived Plasma Exosomes of Alzheimer's Patients*

Diana J. Cha, David Mengel, Maja Mustapic, Wen Liu, Dennis J. Selkoe, Dimitrios Kapogiannis, Douglas Galasko, Robert A. Rissman, David A. Bennett and Dominic M. Walsh

*116 Exosome Production is Key to Neuronal Endosomal Pathway Integrity in Neurodegenerative Diseases*

Paul M. Mathews and Efrat Levy

*130 Intrastriatal Administration of Exosome-Associated Pathological Alpha-Synuclein is Not Sufficient by Itself to Cause Pathology Transmission*

Mantia Karampetsou, Vasia Samantha Sykioti, Emmanouela Leandrou, Katerina Melachroinou, Alexandros Lambiris, Antonis Giannelos, Evangelia Emmanouilidou and Kostas Vekrellis

# Therapeutic Potential of Extracellular Vesicles for the Treatment of Nerve Disorders

Luisa R. Galieva1 , Victoria James 2 , Yana O. Mukhamedshina1,3 \* and Albert A. Rizvanov 1

1 Institute of Fundamental Medicine and Biology, Kazan Federal University, Kazan, Russia, 2 School of Veterinary Medicine and Science, University of Nottingham, Nottingham, United Kingdom, 3 Department of Histology, Cytology, and Embryology, Kazan State Medical University, Kazan, Russia

The use of extracellular vesicles (EVs) as cell free therapy is a promising approach to stimulate tissue regeneration including that of the nervous system. EVs transfer bioactive proteins and lipids, RNA and microRNAs, which play a relevant role in EV-mediated intercellular communication. The immunomodulatory, anti-inflammatory, and neuroprotective effects of mesenchymal stem cells-derived EVs have been well studied, knowledge of this paracrine mechanism and the availability of these cells, positions mesenchymal stem cells as a potential source of EVs for cell free therapy for a variety of regenerative and nervous system disorders. In this review, we focus on the immunomodulatory and neuroprotective effects of stem cells-derived EVs within in vitro and in vivo models of nerve disorders.

#### Edited by:

Grant Thomas Corbett, Harvard Medical School, United States

#### Reviewed by:

#### Susanna Amadio,

Fondazione Santa Lucia (IRCCS), Italy Veronica Perez de la Cruz, Instituto Nacional de Neurología y Neurocirugía (INNN), Mexico

#### \*Correspondence:

Yana O. Mukhamedshina<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Neurodegeneration, a section of the journal Frontiers in Neuroscience

Received: 20 November 2018 Accepted: 12 February 2019 Published: 05 March 2019

#### Citation:

Galieva LR, James V, Mukhamedshina YO and Rizvanov AA (2019) Therapeutic Potential of Extracellular Vesicles for the Treatment of Nerve Disorders. Front. Neurosci. 13:163. doi: 10.3389/fnins.2019.00163 Keywords: extracellular vesicles, immunomodulatory effects, neuroprotective effects, mesenchymal stem cells, nerve disorders

## INTRODUCTION

The need to improve the effectiveness of available therapeutic protocols used within the regenerative medicine field, has led to a number of different approaches. However, the nerve disorders still present a treatment challenge due to the low regenerative potential of the central nervous system (CNS). Stem cell therapy has been at the forefront of regenerative medicine for the last decade, but poor clinical trial reports have shifted the focus of researchers to develop improved protocols and complementary approaches. For example, it became apparent, that the therapeutic effect of stem cells can be enhanced through the expression of genes of neurotrophic factors using gene therapy approaches. Unfortunately, a hazard associated with inducing such genetic changes is the potential for stem cell transformation. Therefore, a critical need still exists for the development of new therapeutic approaches that do not carry the same potential risks.

Confirmation of the paracrine effects of stem cells and the relevance to tissue regeneration was a breakthrough in this area, paving the way for a series of subsequent studies to characterize and leverage this mechanism for therapeutic development. Gnecchi et al. were the first to show that conditioned medium from mesenchymal stem cells (MSCs) overexpressing the gene Akt1 exerted cytoprotective effects on cardiomyocytes exposed to hypoxia in vitro, reduced acute myocardial infarction sizes and improved ventricular functions in vivo (Gnecchi et al., 2005, 2006). Takahashi et al. (2006) similarly found that injection of bone marrow mononuclear cells supernatants increased microvessel density and decreased the fibrotic area within an infarcted heart model, contributing to significant functional improvement. Subsequently, MSCs-derived conditional medium was reported to stimulate functional and morphological improvement in pathological conditions such as renal injury, myocardial infarction, and fulminant hepatic failure (Bi et al., 2007; Parekkadan et al., 2007; Timmers et al., 2007).

The results obtained supported a paracrine hypothesis of stem cell action in tissue protection and repair. It was subsequently established that these beneficial were attributable to soluble factors including extracellular vesicles (EVs) released from stem cells (Bruno et al., 2009; Lai et al., 2010; Xin et al., 2013a). The biogenesis and methodology of isolation of EVs has been described in several published reviews (Biancone et al., 2012; Akers et al., 2013; Koniusz et al., 2016). Therefore, in this mini review, we focus on the newly emerging field investigating the therapeutic potential of stem cell-derived EVs, produced in vitro and transplanted within the animals and patient for the treatment of nerve disorders.

#### IMMUNOMODULATORY AND NEUROPROTECTIVE EFFECTS OF STEM CELL-DERIVED EVS

By transferring bioactive proteins and lipids, RNA and microRNAs (miRNAs), EVs may play a crucial role in intercellular communication between stem cells and other cells within the tissue micro-environment (Camussi et al., 2013; Braccioli et al., 2014; Gomzikova and Rizvanov, 2017). The antiinflammatory and neuroprotective effects of MSC-derived EVs and their paracrine mechanism of action have been well studied (Kalani et al., 2014; Börger et al., 2017).

EVs have been shown to be actively involved in processes of immune regulation such as stimulation of T-cell proliferation, apoptosis induction in activated cytotoxic T-cells, differentiation of monocytes into dendritic cells and B lymphocyte-mediated tumor suppression (Bobrie et al., 2011; Chaput and Théry, 2011; Taylor and Gercel-Taylor, 2011). It is not surprising that MSCs-derived EVs were also found to be immunologically active (Blazquez et al., 2014; Zhang et al., 2014; Bruno et al., 2015). The immunomodulatory properties of MSCs-derived EVs are reported to be the result of the suppression of TNF-a and IL-1b secretion, increasing the concentrations of TGF-b, regulatory T cells (Treg) and cytotoxic T lymphocyte-associated protein 4, inducing Th1 to Th2 cell conversion, reducing IFNγ production and the potential of T cells to differentiate into Th17 cells (Mokarizadeh et al., 2012; Blazquez et al., 2014; Del Fattore et al., 2015; Chen et al., 2016b). However, some data indicate a lower in vitro immunomodulatory effect of MSCderived EVs on T-cell proliferation and antibody formation, as compared with their cellular counterpart (Conforti et al., 2014). In a recent study, immunomodulatory effect inflammationstimulated (TNF-α and IFN-γ) MSCs-derived EVs was evaluated (Harting et al., 2018). It was found, that thus obtained EVs have enhanced anti-inflammatory properties partially due to COX2/PGE2 pathway alteration.

Although EVs can exhibit a variety of different protein and nucleic acid cargos, their regenerative effects are mainly ascribed to the transfer of specific proteins and miRNAs (Collino et al., 2017). For example, EV cargos of miR-1000, miR-133b, miR-21, miR-34a, and miR-219 have been reported to exert a neuroprotective effect by regulating glutamate release at the synapse (Verma et al., 2015), promoting neural plasticity (Xin et al., 2013b), suppressing apoptosis (Ma et al., 2013; Vallabhaneni et al., 2015), and enhancing myelination (Pusic and Kraig, 2014b).

Despite knowledge of the function of some aspects of the EV cargo, further studies are required to fully understand the mechanisms that underpin the immunomodulatory and neuroprotective effects of EVs.

#### CURRENT PERSPECTIVES OF EVS IN THE TREATMENT OF NERVE DISORDERS

Although the therapeutic potential of EVs has not been fully elucidated, their use can be said to be most promising for the treatment of different disorders of the CNS, when drugs are of limited use due to their inability to penetrate the blood-brain barrier (BBB). EVs have been proposed to be transported across the interior of a cell via transcytosis, which may enable these EVs to cross the BBB. This has been experimentally confirmed, as EVs given intravenously (Alvarez-Erviti et al., 2011) or intranasally (Zhuang et al., 2011; Haney et al., 2015) were able to penetrate the BBB.

It should be noted that the therapeutic potential of EVs depends on the composition of their cargo. The cargo of EVs from different cell types can include a common set of biological molecules (nucleic acids, proteins, and lipids) and also molecules that reflect the cell source of the EVs and the physiological or pathological state of the cell source (Zhang et al., 2016; Reiner et al., 2017). In this regard, it is important to define the cargo of stem cell-derived EVs to further understand the mechanisms involved in regenerative processes during therapy. Currently, most studies evaluating the regenerative potential of stem cellderived EVs focus on a general assessment of therapeutic efficacy without detailing the multiple physiological or biochemical changes occurring due to EVs specific cargo. Defining the EV cargo would allow subsequent manipulation, combining a specific set of proteins and RNAs which could efficiently modulate the course of nerve disorders (Selmaj et al., 2017).

The introduction of EV use into clinical practice has been limited by the ability to isolate sufficient EVs from culture systems and the heterogeneity of the cargo of naturally occurring EVs. Therefore, methods of obtaining "modified" EV have become a major focus for the field. In such a cases, EVs are most often derived from MSCs owing to the availability of these cells and the presence of positive properties mediated by their EVs, or induced pluripotent stem cells (iPS cells) whose EVs also demonstrate consistent regenerative abilities. EVs from both cell types have been proposed as a potential starting point for cell free therapy in nerve disorders (**Figure 1**).

**Table 1** provides a summary of preclinical studies of EV use in both in vitro and in vivo models of nerve disorders.

#### Multiple Sclerosis (MS)

The success of remyelinating therapy in MS is mostly attributed to dendritic cell (DCs)-derived EVs (Pusic et al., 2014a), whereby EVs produced by IFN-γ-stimulated DCs increased

myelination and oxidative tolerance in vitro and in vivo (Pusic et al., 2014c). These changes are likely attributable to the high levels of miR-219 within the EVs, which is necessary and sufficient for oligodendrocyte precursor cell (OPC) differentiation, the formation and maintenance of compact myelin, and is deficient in human multiple sclerosis lesions (Junker et al., 2009; Dugas et al., 2010; Zhao et al., 2010). Pusic et al. (2014a) proposed that stimulated DCs-derived EVs contain high levels of specific anti-inflammatory miRNAs that may have an immunomodulatory role in suppressing the development of multiple sclerosis. Yu et al. (2013) demonstrated that EVs with membrane-associated TGF-β1 from gene-modified DCs prevented the de novo differentiation of Th17 cells by inhibiting DC IL-6 production, and were effective in inhibiting the development and progression of experimental autoimmune encephalomyelitis (EAE).

Zhuang et al. (2011) used EVs loaded with anti-inflammatory agents like curcumin or JSI124 for intranasal delivery to the brain. Effects of this route of delivery were evaluated in 3 different inflammation-mediated murine disease models: brain tumorbearing, lipopolysaccharide (LPS)-induced brain inflammation, and EAE. Having given EVs with curcumin, mice were protected from LPS-induced brain inflammation and progression of EAE, brain tumor-bearing mice also showed a significant delay in brain tumor growth following curcumin loaded EV treatment. Intranasal administration of EVs has been shown to provide rapid transport to the brain, and subsequent absorption by the microglia.

A marked reduction in MS and EAE relapses have been observed during pregnancy (Confavreux et al., 1998). It was demonstrated in a mouse model of EAE that serum EVs suppressed T cell activation, promoted the maturation of OPC, and pregnancy-associated exosomes facilitated OPC migration into active CNS lesions (Williams et al., 2013). At the same time, serum EVs derived from non-pregnant mice were also able to reduce the severity of EAE. Thus, it was established that serum EVs are a prominent mediator both of immune modulation and neuroprotection which is especially pronounced during pregnancy.

#### Alzheimer's Disease (AD)

Wang et al. (2017) demonstrated the positive effect of using bone marrow MSC-derived EVs (BM-MSC-derived EVs) both in vitro and in vivo. Inducible nitric oxide synthase (iNOS) mRNA and protein levels were significantly reduced in cultured primary neurons prepared from APP/PS1 pups treated with BM-MSCderived EVs. In vivo studies demonstrated improved cognitive behavior, rescued CA1 synaptic transmission, and long-term potentiation in APP/PS1 mice administrated with BM-MSCderived EVs through an intracerebroventricular (i.c.v.) injection.

It has already been reported, that neutral endopeptidase (NEP) levels and activity are reduced in patients with AD (Yasojima et al., 2001). NEP was initially identified as a regulator of the Aβ level (Iwata et al., 2000). Katsuda et al. (2013) demonstrated NEP-specific enzyme activity could be exhibited by adipose tissue-derived MSC EVs (AD-MSC-derived EVs). Furthermore,

#### TABLE 1 | Preclinical trials using EVs.

(Continued)

TABLE 1 | Continued

it was indicated that AD-MSC-derived EVs when transferred to N2a cells (a fast-growing mouse neuroblastoma cell line overproducing human Aβ) contributed, at least in part, to a decrease in both extracellular and intracellular Aβ levels. That AD-MSCs expressed NEP at a higher level than BM-MSCs is an important observation highlighting the importance of MSC origin on function (Yasojima et al., 2001).

An alternative cellular source of EVs for therapeutic intervention in AD were used by Yuyama et al. (2015). These authors showed that intracerebral infusion of neuronal EVs into brains of APP transgenic mice decreased amyloid depositions, suggesting an important role for neuronal exosomes in clearing Aβ in brain, but not explaining mechanisms of EVs actions.

#### Parkinson's Disease (PD)

Haney et al. (2015) proposed a new EV-based technology for catalase delivery to the CNS to treat PD. Bovine liver derived catalase was loaded into EVs isolated from the mouse macrophage cell line (Raw 264.7). The in vitro study data demonstrated that reactive oxygen species (ROS) levels decreased when catalase-loaded EVs as well as empty ones were co-cultured with cortical neurons and dopaminergic (DA) neurons, although the effect of empty EVs was less significant. Administration of the same catalase loaded EVs to a model of 6-OHDA lesions in C57BL/6 female mice had an anti-inflammatory effect manifested as decreased microgliosis. It is important to note that there was no effect on microglia activation, or the number of DA neurons. It was hypothesized that EVs-mediated delivery of catalase to activated microglia, astrocytes, and neurons in the inflamed brain might result in ROS degradation and neuroprotection in PD patients.

Jarmalaviciute et al. (2015) ˇ demonstrated in vitro that EVs isolated from the supernatants of stem cells derived from the dental pulp of human exfoliated deciduous teeth (SHEDs), and grown on laminin-coated 3D alginate micro-carriers, suppressed 6-OHDA (6-hydroxy-dopamine) induced apoptosis in DA neurons. Interestingly, EVs from the same stem cells cultured under standard conditions possessed no such properties. These results confirm that EVs serve as a novel therapeutic approach in the treatment of Parkinson's disease, although the mechanism of action remains unclear.

#### Amyotrophic Lateral Sclerosis (ALS)

Bonafede et al. (2016) showed in vitro, that AD-stromal cell-derived EVs (AD-SC-derived EVs) were able to protect NSC-34 cells from oxidative damage, which is one of the main mechanisms of damage in ALS, increasing cell viability. They suggest the positive effect might be caused by secretion of miRNAs, which play a protective role via inhibition of apoptosis pathways, and promoting cell cycle progression and proliferation. AD-SC-derived EVs were shown in vitro to ameliorate SOD1 protein levels and aggregates in neuronal cells derived from G93A ALS mice. It was concluded that the abnormal expression of mitochondrial proteins in ALS cells could be rescued by application of AD-SC-derived EVs (Lee et al., 2016).

#### Stroke

When using BM-MSC-derived EVs in a model of middle cerebral artery occlusion (MCAO) ischemic lesion volumes reduced, however there was no significant difference compared to PBStreated controls (Xin et al., 2013a). This study demonstrated that neurite remodeling improved, alongside increases in synaptic plasticity, neurogenesis and angiogenesis within the ischemic boundary zone. These improvements in function were proposed to be the result of miR133b activity on neurite remodeling, delivered to neural cells as part of the BM-MSC-derived EVs cargo (Xin et al., 2013b). In complementary study, intravenous injection of BM-MSC-derived EVs into an in vivo model of MCAO reduced post-ischemic motor coordination impairment, induced long-term neuroprotection, increased cell proliferation, and stimulated neurogenesis and angiogenesis (Doeppner et al., 2015).

Chen et al. (2016a) studied the effects of intravenous administration of AD-MSC-derived EVs and AD-MSCs in a model of MCAO. Results demonstrated that neurological impairment was reversed after treatment with AD-MSC-derived EVs an effect that was more significant when treatment used AD-MSCs. Reduced infiltration of CD11+ and CD68+ cells (two indicators of inflammation), decreased oxidative stress and increased angiogenesis were reported in both experimental groups.

Otero-Ortega et al. (2017) demonstrated in a model of subcortical stroke that after intravenous infusion, EVs were found in the brain and peripheral organs (the liver, lungs and the spleen). There was improved functional outcome, increased axonal sprouting, increased oligodendrocyte-associated marker expression and myelin formation, changes in white matter thickness and the restoration of tract connectivity at 28 days after EV administration. Proteomics analysis of the EVs identified 2,416 proteins that are implicated in repairing brain functions.

#### Neurotrauma

Zhang et al. (2015) demonstrated that BM-MSC-derived EVs effectively improved functional outcome by promoting endogenous angiogenesis, neurogenesis and reducing inflammation in rats after traumatic brain injury (TBI). In 2 years the same team of scientists reported the results of systemic administration of BM-MSC-derived EVs cultured under 2D and 3D conditions. This treatment did not alter cortical lesion volume but significantly improved cognitive and sensorimotor functional recovery, increased the number of newly formed mature neurons in the dentate gyrus (DG). It also increased the number of newly formed endothelial cells in the lesion boundary zone and DG, as well as reduced neuroinflammation. BM-MSC-derived EVs cultured under 3D conditions provided better outcome in spatial learning compared to EVs from a 2D culture (Zhang et al., 2017).

Kim et al. (2016) demonstrated that BM-MSC-derived EVs suppressed neuroinflammation after TBI in mice. It was also shown that an intravenous infusion of the isolated EVs shortly after induction of TBI rescued pattern separation and spatial learning impairments 1 month later.

Studies of the therapeutic potential of EVs in spinal cord injury (SCI) report that systemic administration of BM-MSC-derived EVs in contused SCI in rats attenuated cell apoptosis and inflammatory processes, stimulated angiogenesis, and promoted functional recovery. In particular, there was a downregulated expression of a pro-apoptotic protein (Bax) and pro-inflammatory cytokines (TNF-α and IL−1β), increased expression levels of anti-apoptotic (Bcl-2) and anti-inflammatory (IL-10) proteins (Huang et al., 2017). Furthermore, Lankford et al. (2018) demonstrated that intravenously delivered BM-MSC-derived EVs targeted the SCI site, might contribute to the therapeutic effects by specifically targeting M2 macrophages. EVs were detected in the area of SCI and the spleen, but not in the intact region of the spinal cord.

Recently, Wang et al. (2018) confirmed a comparable therapeutic effect of intravenously administered BM-MSCderived EVs and BM-MSCs in SCI. The authors observed that BM-MSC-derived EVs reduced the proportion of neurotoxic astrocytes, probably via inhibiting nuclear translocation of NFκB p65, and exerted anti-inflammatory and neuroprotective effects following SCI. Similar results were obtained by Ruppert et al. (2018), who confirmed the anti-inflammatory and functional effects of intravenously delivered BM-MSC-derived EVs by means of a decreased in microglia activation markers rather than skewing microglial polarization.

#### Clinical Trials Using EVS in the Treatment of Nerve Disorders

The conceptual advance in our understanding of the therapeutic application of EVs as a regenerative strategy for neural tissue is relatively new. Therefore, a considerable number of studies are non-clinical, relying on in vitro and in vivo models. To date the Food and Drug Administration (FDA) have approved clinical trials using EVs as a treatment of ulcers (ClinicalTrials.gov Identifier: NCT02565264), sepsis (NCT02957279), macular hole (NCT03437759), and type 1 diabetes mellitus (NCT02138331). Based on pre-clinical studies of EV-mediated delivery of miR-124 promoting neurogenesis after ischemia (Yang et al., 2017), a further clinical trial using MSC-derived EVs in the treatment of acute ischemic stroke (NCT03384433) is planned for 2018–19, in which the effect of MSC-generated EVs will be examined in post-stroke phases 1 and 2.

## CONCLUSION

Studies of the therapeutic potential of EVs in regenerative strategies strongly support the use of MSC-derived EVs in the treatment of nerve tissue disorders. MSC-derived EVs are reported to exert the majority of the positive effects seen with the direct use of MSCs as a therapy, but without the risks associated with stem cell transplantation (Gatti et al., 2011; Li et al., 2012; Konala et al., 2016). The rapid progression from non-clinical to clinical studies of MSC-derived EVs will uncover the exciting potential that EV-mediated cell free therapy may offer for the treatment of nerve disorders.

## AUTHOR CONTRIBUTIONS

LG: collection of data on the characteristics of immunomodulatory and neuroprotective effects of EVs,

#### REFERENCES

compilation these data. VJ: collection of data on the transplantation EVs in neurodegeneration deseases, professional English editing. YM: collection of data on the transplantation EVs in stroke and traumatic injury, compilation these data, drawing **Figure 1**. AR: compilation of article content, writing some chapters.

## FUNDING

This work was supported by a grant from the Russian Science Foundation No. 18-75-00043 YM. This work was performed in accordance with the Program of Competitive Growth of the Kazan Federal University and a subsidy allocated to the Kazan Federal University for the state assignment in the sphere of scientific activities. AR was supported by a state assignment 20.5175.2017/6.7 (Leading Researcher) of the Ministry of Education and Science of the Russian Federation.

mesenchymal stromal cells on functional recovery and neurovascular plasticity in rats after traumatic brain injury. J. Neurosurg. 122, 856–867. doi: 10.3171/2014.11.JNS14770

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Galieva, James, Mukhamedshina and Rizvanov. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Leukocyte Derived Microvesicles as Disease Progression Biomarkers in Slow Progressing Amyotrophic Lateral Sclerosis Patients

Daisy Sproviero1† , Sabrina La Salvia1† , Federico Colombo2 , Susanna Zucca1 , Orietta Pansarasa1 , Luca Diamanti3,4, Alfredo Costa3,4, Luca Lova3,5, Marta Giannini1,3 , Stella Gagliardi1 , Eliana Lauranzano6 , Michela Matteoli6,7, Mauro Ceroni3,4 , Andrea Malaspina8† and Cristina Cereda1 \* †

1 Genomic and Post-Genomic Center, IRCCS Mondino Foundation, Pavia, Italy, 2 Flow Cytometry and Cell Sorting Unit, Humanitas Clinical and Research Center – IRCCS, Rozzano, Italy, 3 Department of Brain and Behavioral Sciences, University of Pavia, Pavia, Italy, 4 Division of General Neurology, IRCCS Mondino Foundation, Pavia, Italy, 5 Becton Dickinson Italia S.p.A., Milan, Italy, 6 Laboratory of Pharmacology and Brain Pathology, Humanitas Clinical and Research Center – IRCCS, Rozzano, Italy, 7 IN-CNR, Milan, Italy, 8 Neurodegeneration Group, Centre for Neuroscience and Trauma, Blizard Institute, Queen Mary University of London, London, United Kingdom

#### Edited by:

Grant Thomas Corbett, Harvard Medical School, United States

#### Reviewed by:

Bradley Turner, The Florey Institute of Neuroscience and Mental Health, Australia Gérard Lizard, Université de Bourgogne, France

#### \*Correspondence:

Cristina Cereda<EMAIL_ADDRESS>†These authors have contributed equally to this work

#### Specialty section:

This article was submitted to Neurodegeneration, a section of the journal Frontiers in Neuroscience

Received: 20 December 2018 Accepted: 25 March 2019 Published: 15 April 2019

#### Citation:

Sproviero D, La Salvia S, Colombo F, Zucca S, Pansarasa O, Diamanti L, Costa A, Lova L, Giannini M, Gagliardi S, Lauranzano E, Matteoli M, Ceroni M, Malaspina A and Cereda C (2019) Leukocyte Derived Microvesicles as Disease Progression Biomarkers in Slow Progressing Amyotrophic Lateral Sclerosis Patients. Front. Neurosci. 13:344. doi: 10.3389/fnins.2019.00344 The lack of biomarkers in Amyotrophic Lateral Sclerosis (ALS) makes it difficult to determine the stage of the disease in patients and, therefore, it delays therapeutic trials. Microvesicles (MVs) are possible biomarkers implicated in physiological and pathological functions, however, their role in ALS remains unclear. We investigated whether plasma derived microvesicles could be overrepresented in a group of 40 patients affected by ALS compared to 28 Alzheimer's Disease (AD) patients and 36 healthy volunteers. Leukocyte derived MVs (LMVs) compared to endothelial, platelet, erythrocyte derived MVs, were mostly present in ALS patients compared to AD patients and healthy donors. Correlation analysis corrected for the presence of confounding variables (riluzole, age at onset, site of onset, gender) was tested between PRL (Progression Rate at the Last visit) and LMVs, and a statistically significant value was found (Pearson partial correlation r = 0.407, p = 0.006). We also investigated SOD1, TDP-43 intravesicular protein level in LMVs. Misfolded SOD1 was selectively transported by LMVs and its protein level was associated with the percentage of LMVs in slow progressing patients (r = 0.545, p = 0.033). Our preliminary findings suggest that LMVs are upregulated in ALS patients and they can be considered possible markers of disease progression.

Keywords: amyotrophic lateral sclerosis, biomarkers, disease progression, microvesicles, SOD1, TDP-43

## INTRODUCTION

The discovery of disease biomarkers for prognostic purposes, clinical monitoring, and evaluation of treatment response is a major research endeavor in Amyotrophic Lateral Sclerosis (ALS), a fatal neurodegenerative disease caused by selective motor neuron death (Al-Chalabi and Hardiman, 2013; Al-Chalabi et al., 2016). Microvesicles (MVs), a subclass of

**Abbreviations:** ALS, amyotrophic lateral sclerosis; EVs, extracellular vesicles; LMVs leukocyte derived microvesicles; MVs, microvesicles.

extracellular vesicles, are biologically relevant, considering their cargo of RNAs, proteins, and surface receptors and they have potential to be used as biomarkers in both physiological and pathological states (Cocucci and Meldolesi, 2015). MVs (size: 100–1000 nm) are vesicles shed by budding of the plasma membrane of cells (Raposo and Stoorvogel, 2013; Lötvall et al., 2014; Cocucci and Meldolesi, 2015). They are present under physiological conditions, but they can be significantly elevated under various stimuli [increased (Ca2+), cellular stress, cytokine exposure, etc.] and in pathological conditions, such as cancer and neurodegenerative diseases (Yáñez-Mó et al., 2015). Upon release from their cell of origin, MVs interact only with cells that they recognize for their receptors and, once recognized, they can fuse with the plasma membrane and discharge their cargo into the cytoplasm (Prada and Meldolesi, 2016). MVs have a central role in inflammatory processes (Carandini et al., 2015) and they could be plausible targets in any research into ALS, which is characterized by an activation of astrocytes and microglia (Henkel et al., 2004; Cereda et al., 2008) as an immunological reaction to motor neuron death. Immune responses can be triggered by pathological proteins, like SOD1 and TDP-43, which have relevance to neurodegeneration (Cereda et al., 2013; Amor et al., 2014). Misfolded SOD1 is able to activate microglia by binding to the CD14/TLR4 receptor (Beers et al., 2008) and expression of TDP-43 increases proinflammatory markers, like IL-6, and TNFa which in glial and neuronal cells from ALS patients can act as co-activators of NFκB (Swarup et al., 2011). SOD1 and TDP-43 can be transported by extracellular vesicles (Feneberg et al., 2014; Hanspal et al., 2017; Sproviero et al., 2018), however, it is not known whether MVs protein cargo contributes to the progression of ALS pathology. We have previously demonstrated that plasma derived MVs of ALS patients were enriched with SOD1, TDP-43, and FUS compared to controls, but we didn't investigate if these proteins were transported by MVs of a specific origin. Zachau's group has previously demonstrated the presence of high level of LMVs (Leukocytes derived microvesicles-CD45+MVs) in the cerebrospinal fluid (CSF) of an ALS patient (Zachau et al., 2012). Here, we investigated the role of CD45 MVs sub-typing in blood for clinical stratification of ALS patients and MVs function, as potential carrier of misfolded proteins, alternative route for disease propagation.

#### MATERIALS AND METHODS

#### Standard Protocol Approvals, Registrations, and Patient Consents

The study protocol was approved by the Ethical Committee of the IRCCS Mondino Foundation (Pavia, Italy). Subjects participating in the study signed an informed consent (Protocol n◦ 375/04 – version 07/01/2004). The study conformed the standards of the Declaration of Helsinki. Plasma was isolated from 40 sporadic ALS patients (SALS) (mean age at sampling: 67 ± 9.91). ALS diagnosis was made according to the revised El Escorial Criteria (Brooks et al., 2000). ALS individuals harboring mutations in the SOD1, FUS/TLS, TARDBP, C9ORF72 were excluded from this study. Patients with concomitant infections (pneumonia and infection at the site of gastrostomy) were excluded and biological signs of inflammation present (CRP-ESR) were normal. Patients' demographic and clinical characteristics are reported in **Table 1**. Progression rate at the last visit (PRL) was calculated as 48 minus the ALS Functional Rating Scale–Revised score (ALSFRS) at the last visit divided by the disease duration (in months) from onset of symptoms to the last visit (48-ALSFRS/1t) (Lu et al., 2015). Progression rate lower than 0.5, and higher than 0.5 were defined as slow (ALS-slow) and fast progressing ALS (ALS-fast), respectively. Thirty-six sex and age-matched healthy volunteers, not on any pharmacological treatment (mean age: 51.04 ± 9.9) were used as controls (CTRL). Plasma was also collected from 28 patients affected by Alzheimer's Disease (AD; mean age at sampling: 75.8 ± 7.3). Diagnosis of AD was based on Aging-Alzheimer's Association work group criteria (McKhann et al., 2011). AD patients were used as neurological controls to assay if the evaluated parameters are specific of the disease. All methods were performed in accordance with the relevant guidelines and regulations.

#### Blood Sample Collection and Isolation of MVs

Blood samples were obtained from patients with ALS and AD and healthy controls by peripheral venepuncture into BD VacutainerTM blood collection tubes with Sodium Citrate (BD Biosciences, United States). Within 1 h, blood sample was centrifuged at low speed (1,000 × g for 20 min, 1600 × g for 15 min) to separate plasma and remove platelets. Platelet-free plasma was then transferred to a new tube and snap frozen at −80◦C. Prior to the analysis, platelet-free plasma was thawed on ice and it was centrifuged at 20,000 × g for 1 h with Centrifuge 5427R (Eppendorf, Italy). The pellet was washed with 0.22 µm filtered PBS and centrifuged at 20,000 × g for 1 h. The pellet was then processed for MVs analysis. Western Blot analysis for MVs marker (Annexin V, Santa Cruz Biotechnology, United States) and EXOs marker (Alix, Abcam, United States) and Nanoparticle-tracking analysis (NTA) were run to confirm MVs purity (**Supplementary Figure S1**) as previously described (Sproviero et al., 2018).

#### Flow Cytometry Analysis of MVs

Microvesicles pellet was re-suspended in 1 ml of 0.22 µm filtered Annexin V binding buffer 1× (BD Biosciences, United States). MVs were incubated with conjugated primary antibody as listed

†ALSFRS, ALS Functional Rating Scale; PRL, Progression Rate at last visit.

in the antibody section. Samples were analyzed immediately after labeling, using a BD FACS Canto II with BD FACS Diva software (BD Biosciences, United States). A standardized calibrated-bead strategy using polystyrene beads (Megamix-Plus, BioCytex, France) was used, as previously described (Nielsen et al., 2014), to discriminate MVs from background noise. The polystyrene/latex beads used were a mix of fluorescent beads of varied diameters, selected to cover a theoretical MVs size range (0.16–0.20 and 0.2–0.5 µm), using SSC as a size-related parameter. Polystyrene/latex beads acquisition setting allows the cytometer to study MVs within a constant size region and getting reproducible MV counts. The range 0.16–0.20 µm was used to define the threshold background and MVs range was between 0.2 and 0.5 µm as shown in **Supplementary Figure S1**. MVs with positive staining for Annexin V and cell specific markers CD45 (leukocyte antigen), CD31 (endothelial cell antigen), CD61 (platelet antigen), and CD235a (erythrocyte antigen) were selected. Logarithmic amplification was used for all channels and results were referred to the percentage of Annexin V+ MVs, co-expressing another specific cell lineage marker, as previously described in the literature (Robert et al., 2009).

### Immunoprecipitation of Leukocyte Derived Microvesicles (LMVs)

Microvesicles were separated as described above and resuspended in PBS+BSA 1%. Anti-CD45 antibody (Santa Cruz Biotechnology, United States) was coupled to Dynabeads (Invitrogen, United States) and then incubated with MVs overnight at 4◦C. Proteins retained (immunoprecipitated, IP) on the beads were recovered by adding cold Radio-Immunoprecipitation Assay (RIPA) buffer containing phosphatase and protease inhibitors (Sigma-Aldrich, Italy) and Laemmli buffer 2× and boiled at 95◦C for 5 min to obtain CD45+ MVs. Immunodepleted fraction (I-) and input (starting material of MVs) were lysed in cold RIPA buffer, mixed with Laemmli buffer 2× and denatured at 95◦C for 10 min.

#### Western Blot Analysis

Proteins were fractionated by size on SDS Precast 8–16% polyacrylamide gels (BioRad, Italy), transferred to a nitrocellulose membrane using a Trans-blot Turbo (BioRad, Italy) and blocked with blocking solution (5% non-fat dry milk in Tween-20 Tris-Buffered Saline solution, TBS-T) for 1 h. Membranes were incubated overnight with primary antibody in blocking solution at 4◦C. Membranes were then incubated for 1 h at room temperature with secondary antibodies. Antibodies used are listed in the antibodies section. Bands were visualized using an enhanced chemiluminescence detection kit (ECL Advance, GE Healthcare, United Kingdom). For subsequent immunoreactions, primary and secondary antibodies were removed from the membrane with stripping solution incubated for 20 min (100 mM Glycine, 0.1% NP-40, 1% SDS pH 2.2). Densitometric analysis of the bands was performed using ImageJ software (National Institutes of Health, United States).

## Machine Learning Analysis

Orange software (Demsar et al., 2013) was used to perform an exploratory analysis of MVs distribution in CTRL and ALS groups, with a focus on the percentages of Annexin V+ MVs expressing specific cell lineage markers. Logistic regression was performed and receiver operator characteristic (ROC) curves were computed (Cook, 2008). The percentages of Annexin V+ MVs expressing the cell lineage markers (CD45+/Annexin V+, CD235a+/Annexin V+, CD31+/Annexin V+, CD61+/Annexin V +), were used as regressors for the multivariate analysis and referred to disease phenotypes as described in flow cytometry analysis. Briefly, multivariate logistic regression was performed to predict the class of each sample (healthy donors or patients) and to rank features based on mutual information criteria (Peng et al., 2002).

## Antibodies

Antibodies, used for flow cytometry were mouse monoclonal anti-human CD45 (2D1) (ab 641417APC-H7); mouse monoclonal anti-human CD31 (MEC13.3) (ab 560983 PE); mouse monoclonal anti-human CD61 (RUU-PL7F12) (ab 347408 Per-cy); mouse monoclonal anti-human CD235a (GA-R2 (HIR2) (ab 563666 Pe-cy7) derivation and the apoptotic marker Annexin V (ab 550407 APC-BD) (BD Biosciences, United States). Immunoglobulin isotype-matched control antibodies were purchased from BD Biosciences, United States. Antibodies used for western blot were rabbit polyclonal primary antibody anti-CD45 (Santa Cruz Biotechnology, United States), mouse monoclonal anti-misfolded SOD1-DSE2-3H1 (kindly given by Prof. Neil R. Cashman) and mouse monoclonal anti-TDP-43 (Proteintech, United States). For phospho-TDP-43 we used the same antibody we utilized for the detection of TDP-43, which recognizes the intact 45 kDa protein as well as all post-translationally modified (phosphorylated and glycosylated forms) and truncated forms in multiple applications. Secondary antibodies used were donkey anti-rabbit or anti-mouse secondary peroxidase-conjugated antibody (GE Healthcare, United Kingdom).

## Statistical Analysis

The statistical analysis was carried out using Graph Pad Prism 5.0 (GraphPad Inc., United States) and SPSS statistical package version 22 (IBM, United States). One-way ANOVA with Bonferroni's multiple comparison test was used and a value of p < 0.05 was considered significant. The Shapiro–Wilk test was used to test variables for normality distribution and Levene test was used to test the assumption of homogeneity. In order to determine the correlation between continuous variables, a Pearson's correlation test was applied when the variables were normally distributed and a Spearman's correlation test when the variables were not normally distributed. Categorical variables were tested using the non-parametric Mann–Whitney U-test. A partial correlation analysis (Pearson, one-tailed test) was applied to test the correlation between PRL and CD45, while correcting for the presence of confounding variables such as riluzole (Calvo et al., 2017) or other significant factors (age at

onset, site of onset, gender). A Log-transformation was used for some data if they were not normally distributed.

#### RESULTS

#### LMVs Are Elevated in Plasma of ALS Patients

Correct purification of MVs was confirmed by transmission electron microscopy (TEM), NTA and the detection of classical MVs enriched markers (**Supplementary Figures S1A–C**) as we previously described (Sproviero et al., 2018). In Sproviero et al. (2018), we demonstrated that 90% of MVs were smaller than 500 nm by NTA. For this reason we used specific polystyrene/latex beads in the range of 0.2–0.5 µm. The percentages of Annexin V+ MVs derived from platelets (CD61), erythrocytes (CD235a), leukocytes (CD45), and endothelium (CD31) were analyzed by flow cytometry. AD patients were used as neurological controls to assay if the evaluated parameters are specific of the disease. Annexin V, that binds phosphatidylserine, is used as common marker for MVs (Lötvall et al., 2014). Flow cytometry dot plots show higher levels of LMVs in ALS patients (**Figures 1A,B,D**). Specifically, we observed that 75% of ALS patients had higher levels of leukocyte derived MVs (LMVs, % CD45+/Annexin V+ MVs) as percentage of all MVs subsets than the mean of the non-neurological control individuals and AD patients considered in this study as neurological controls (**Figure 1C**, ANOVA followed by Bonferroni's test, p < 0.0001; p < 0.001, respectively). Conversely, percentages of Annexin V+ MVs derived from endothelium (CD31), erythrocytes (CD235a), and platelets (CD61) percentage (**Figures 1E–G**) of ALS patients were not different from those of healthy control subjects and AD patients. A Spearman correlation test was run among the four markers and the age (**Table 2**). Even if there was a significant correlation between age and CD45 MVs %, the data of the three groups didn't meet the assumption of homogeneity of regression slopes since the Levene test was significant (F = 16.47, p-value < 0.001), so we didn't correct for the age.

Multivariate logistic regression analysis (MLRA) was applied as a classification method for ALS and control data for the four markers (**Figures 2A–D**). CD45+/Annexin V+ MVs were identified as the most informative feature to discriminate ALS subgroup from AD patients and healthy controls (**Figure 2A**). The ROC curve (**Figure 2E**) showed an area under the curve of 0.717 and accuracy of 0.653 in separating patients from healthy controls. In ALS patients, no difference was found in LMV% between male and female patients (Mann–Whitney U-test p = 0.967) (**Table 3**). There was no statistically significant

FIGURE 1 | LMVs overrepresentation in plasma from ALS patients. Flow cytometry dot plots of MVs isolated from plasma of a representative healthy control, an ALS patient and AD patient labeled with Annexin V and CD45 (A–C). While healthy control (A) and AD patient (C) presented few events similar to isotype control, ALS patient (B) had higher % CD45+/Annexin V+ MVs. Dot plots of MVs isolated from plasma of ALS patients (red), healthy controls (green), and AD patients (blue) labeled with Annexin V and CD45 (D), CD31 (E), CD235A (F), CD61 (G) markers. Results were referred as the percentage of Annexin V+ MVs expressing the cell lineage marker. While Annexin V+ MVs derived from endothelium (%CD31 MVs) (E), erythrocytes (%CD235a MVs) (F), platelets (%CD61 MVs) (G) were not different from the healthy control subjects and AD patients (ANOVA test, p > 0.05), the percentage of Annexin V+ MVs derived from leukocytes (%CD45 MVs) (D) was significantly enhanced in the plasma of ALS patients compared to the control group (ANOVA test, ∗∗∗p < 0.001) and the AD patients (ANOVA test, ∗∗p < 0.01). n CTRL = 36; n ALS = 40; n AD = 28.

TABLE 2 | Spearman correlation between CD45, CD235a, CD31, CD61% MVs, and age.

§,∗Significant level, p-value < 0.05; ∗∗Significant level, p-value < 0.01, ∗∗∗Significant level, p-value < 0.001.

correlation between age at onset and CD45 MVs (Spearman test, r = 0.370, p = 0.072) (**Table 3**).

#### LMVs Selective Enrichment of Misfolded SOD1

We have looked at whether LMVs (the most represented MVs in blood from ALS patients), carry different levels of misfolded SOD1 and TDP-43 proteins. LMVs (CD45+MVs) were isolated from plasma of 19 ALS patients (13 = Slow; 6 = Fast) and 10 healthy controls. LMVs of all patients analyzed were enriched with misfolded SOD1 and densitometric analysis of this protein, normalized against CD45, revealed a slight increase of this protein in ALS patients compared to controls (0.8431 ± 0.1236 for CTRL and 1.008 ± 0.1274 for ALS) (**Figure 3B**). The immunodepleted fraction (I-), which includes all CD45 negative MVs derived from other cells, did not show any or very little level of misfolded SOD1 (**Figure 3A**), found mainly in LMVs.

Leukocyte derived MVs of only 12 out of 19 patients carried a TDP-43 band at 45 kDa, which resembles the phosphorylated form of TDP-43 (p-TDP-43) (Steinacker et al., 2008). TDP-43 was found in IP fraction (CD45+MVs) and in the immunodepleted (I-) fraction (CD45−MVs) (**Figure 3A**). These results indicate that misfolded SOD1, but not TDP-43, may be compartmentalized in LMVs.

#### LMVs Correlation With Rate of Disease Progression and Misfolded SOD1 Enrichment

A Pearson partial correlation between LMVs and PRL was evaluated, since the variables were normally distributed. The

†Mann–Whitney U-test; §Spearman's correlation test; ∗Significant level; p-value < 0.05.

FIGURE 3 | Misfolded SOD1 enrichment in LMVs (CD45+ MVs) from ALS patients and controls. CD45+ MVs of patients (ALS 1 and ALS 2) and of CTRLs (CTRL1) were enriched with misfolded SOD1. Misfolded SOD1 was only found in LMVs. The cropped blots are used in the figure and full length blots are presented in Supplementary Figure S2. The immunodepleted (I-) fraction, which include all CD45 negative MVs from other cell origin, showed no or very little level of misfolded SOD1. TDP-43 was transported in its phosphorylated form by MVs of the IP and immunodepleted (I-) (A). ALS 1 and ALS 2 are referred to two slow progressing patients. Input = MVs whole lysate-20% of the IP; IP = CD45 immunoprecipitated MVs (LMVs); I- = immunodepleted. (B) Densitometric analysis of misfolded SOD1, normalized to CD45 band, revealed slight increase of misfolded SOD1 in all ALS patients compared to controls (0.8431 ± 0.1236, CTRL n = 10; 1.008 ± 0.1274, ALS n = 18).

FIGURE 4 | LMVs percentage in ALS patients is correlated to progression rate at last visit and is correlated to misfolded SOD1 protein level in slow progressing ALS patients. The log of PRL and of LMVs was plotted and the line was drawn from the least squares regression. A significant correlation was found between LMVs and PRL (Pearson partial correlation r = 0.409, p = 0.006) (A). The PRL of slow progressing patients was positively correlated to LMVs levels (% CD45 MVs) (Pearson partial correlation r = 0.432, p = 0.022) (B). On the other hand, there was no correlation between LMVs levels and disease progression in fast progressing patients (Pearson partial correlation, r = –0.0037, p = 0.496) (C). CD45+ MVs percentage (%) was directly correlated with misfolded SOD1. Densitometric analysis of misfolded SOD1 was correlated to the percentage of CD45 Annexin V (%CD45 MVs) by Spearman rank analysis. LMVs level was not associated to the densitometric level of misfolded SOD1 in ALS patients (Spearman test, r = 0.288, p = 0.123) (D). We instead found a strong correlation between misfolded SOD1 protein level and LMVs in slow progressing patients (r = 0.545, p = 0.033) (n = 12) (E). No statistically significant difference was found in fast progressing patients (r = –0.371, p = 0.234) (n = 6) (F).

presence of a patient with a very fast progression was observed and excluded from the analysis (ALS 20, PRL = 3.12, >3sd from the mean of the PRL distribution). The cohort of patients included a number of six cases that were treated with riluzole medication, eight cases with bulbar onset and 32 with spinal onset. The effect of riluzole and site of onset on PRL were

considered and corrected. Other variables were tested, but resulted not statistically significant (**Table 3**). A significant correlation was found between LMVs and PRL (Pearson partial correlation, r = 0.409, p = 0.006) (**Figure 4A**). The inclusion of the outlier in the correlation analysis did not change the results (Pearson partial correlation, r = 0.379, p = 0.009).

Amyotrophic Lateral Sclerosis patients were separated according to PRL, into slow progressing (PRL < 0.5; n = 23) versus fast progressing (PRL > 0.5; n = 17). The PRL of slow progressing patients was positively correlated to LMVs levels (% CD45 MVs) (r = 0.445, p = 0.022) (**Figure 4B**). On the other hand, there was no correlation between LMVs levels and disease progression in fast progressing patients (r = −0.0037, p = 0.496) (**Figure 4C**). The correlation between the percentage of LMVs and misfolded SOD1 was tested for 19 patients. After removing an outlier for SOD1 (>3sd from distribution mean), LMVs level was not associated to the densitometric level of misfolded SOD1 in ALS patients (Spearman test, r = 0.288, p = 0.123) (**Figure 4D**). We instead found a strong correlation between misfolded SOD1 protein level and LMVs in slow progressing patients (r = 0.545, p = 0.033) (n = 12) (**Figure 4E**). No statistically significant difference was found in fast progressing patients (r = −0.371, p = 0.234) (n = 6). We also found no correlation between LMVs and p-TDP-43 (r = −0.371, p = 0.146) (**Figure 4F**).

#### DISCUSSION

Microvesicles are important mediators of cross-talk among cells and are emerging as new biomarkers of neurological diseases. In this study, for the first time, we have identified an over-representation of the blood CD45+ MVs (LMVs) component in ALS patients compared to healthy controls and to AD patients. Adding to previous observations of an enrichment of CD45+ MVs in CSF from ALS patients (Zachau et al., 2012), we are hinting at LMVs as readout of innate immune response activation at a systemic level in ALS patients. This observation is relevant considering the central role that MVs occupy in any endeavor to develop biomarkers of neurodegeneration.

The LMVs may originate from neutrophils, monocytes or macrophages, lymphocytes, and can also derive from T-cells activation (Zachau et al., 2012). T-regulatory lymphocytes (Tregs) are important immunomodulatory cells that regulate the balance between activation and suppression of the immune response and control microglia activation in the central nervous system. The involvement of T-cells in ALS is supported by a host of experimental data. Treg levels in peripheral blood of ALS patients are inversely correlated with disease severity (Henkel et al., 2013) and it has been shown that rapid disease progression in ALS is associated with a higher number of differentially expressed genes in monocytes (Zhao et al., 2017).

We have found that LMVs blood expression variably correlate to PRL in ALS individuals. LMVs peripheral expression is positively linked to a slower disease progression, but not with fast disease progression. This might depend on the reported Tregs (suppressor T cells) regulation, which modulate the immune system in slowly progressing patients (Henkel et al., 2013; Murdock et al., 2016).

In ALS, extracellular vesicles have been implicated in the release and uptake of misfolded proteins mainly in vitro cell culture (Gomes et al., 2007; Nonaka et al., 2013; Grad et al., 2014; Iguchi et al., 2016; Pinto et al., 2017). SOD1 misfolding has been reported in all ALS sub-types, including the non-SOD1-linked familial and sporadic cases (Shibata et al., 1994) and several studies propose that misfolded protein can be both secreted and taken up by the extracellular environment through exosomes (Gomes et al., 2007; Nonaka et al., 2013; Grad et al., 2014; Iguchi et al., 2016; Pinto et al., 2017).

CD45 can bind to heparan sulfate proteoglycans (Altin and Sloan, 1997), which have been shown to be involved in aggregate uptake of prion proteins (Coombe et al., 1994; Holmes et al., 2013), and therefore, this family of receptors can be involved in the propagation of misfolded SOD1 (Gomes et al., 2007; Grad et al., 2014). In this study, we show that LMVs (CD45+ MVs) selectively transport misfolded SOD1 (but not TDP-43) and that there is a strong correlation between misfolded SOD1 protein levels in LMVs mainly in slow progressing ALS patients. This finding may suggest that slow progression may be linked to the removal of potentially toxic proteins in MVs from the blood stream. Hence LMVs may have a neuroprotective role acting as physiological scavengers of misfolded SOD1 in slow progressing ALS patients as much as in healthy controls.

We have also found p-TDP-43 in LMVs from patients, but not specifically in LMVs. Pathological TDP-43 is hyperphosphorylated and abnormally cleaved to generate aggregationprone C-terminal fragments (CTFs) and it can propagate from cell to cell as p-TDP-43 aggregates (Lagier-Tourenne et al., 2010). However, in our study levels of LMVs were not correlated to TDP-43, in line with previous reports. TDP-43 has been previously reported in secreted exosomes from Neuro2a cells and primary neurons, but not in exosomes produced by CD45 positive astrocytes or microglia (Iguchi et al., 2016). Our data suggest that the regulation of LMVs formation may be key to the immune response to neurodegeneration, exerting a neuroprotective effect by SOD1 misfolded protein removal, impacting on speed of disease progression. These findings might open new avenues for biomarkers discovery and strategies for therapeutics, which enhance aggregated protein clearance.

#### ETHICS STATEMENT

The study protocol was approved by the Ethical Committee of the IRCCS Mondino Foundation (Pavia, Italy). Subjects participating in the study signed an informed consent (Protocol n◦ 375/04 – version 07/01/2004). The study conformed the standards of the Declaration of Helsinki.

#### AUTHOR CONTRIBUTIONS

fnins-13-00344 April 15, 2019 Time: 15:33 # 8

DS executed the experiments, designed the study, acquired and analyzed the data, drafted the manuscript, and created the figures. SLS executed the experiments, and acquired and analyzed the data. FC and LL executed the flow cytometry experiments, and acquired and analyzed the data. SZ performed the machine learning analysis and statistical analysis. OP acquired and analyzed the data. LD and MC recruited the ALS patients and analyzed the data. AC recruited the AD patients and analyzed the data. LL, SG, and EL analyzed the data. MG acquired the data. MM and AM analyzed the data and drafted the manuscript. CC conceived and designed the study, analyzed the data, and drafted the manuscript.

#### FUNDING

This work was supported by Italian Ministry of Health (Grant No. RC2017-2019). AriSLA Foundation for funding (Granulopathy-VCP and autophagolysosomal pathway: guardians of proteostasis and stress granule dynamics. Unraveling their implication in ALS); Fondazione Regionale per la Ricerca Biomedica for TRANS–ALS (Translating Molecular Mechanisms into ALS risk and patient's well-being: FRRB 2015-0023); European Union's Horizon 2020 Research and Innovation Program under grant agreement N◦ 643417 (Grant ID: 01ED1601A, CureALS).

#### REFERENCES

#### ACKNOWLEDGMENTS

We thank Prof. Neil R. Cashman for the gift of the antibody DSE2-3H1. We also thank patients and families for contributing to this study.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fnins. 2019.00344/full#supplementary-material

FIGURE S1 | Microvesicles isolation. (A) Representative image of MVs (2 MVs of about 150 nm) from plasma obtained by transmission electron microscopy (TEM) (Scale bar: 100 nm); (B) Nanoparticle distribution by NTA confirmed the purity of MVs (mode = 137.8 +/− 6.0 nm) (C) Western Blot of MVs and EXOs markers in MVs and EXOs samples showed the presence of Annexin V in MVs lysate and Alix in EXOs lysate.

FIGURE S2 | Western blot analysis of co-immunoprecipitation of CD45+ MVs with misfolded SOD1 and TDP-43 of Figure 3A. Immunoblots of MVs (input), immunoprecipitated CD45+ MVs (Bound) and of CD45- MVs (Unbound) from plasma of an healthy donor (CTRL1-lane 1, 4, 7) and two ALS patients (ALS 1-lane 2, 5, 8) and ALS 2-lane 3, 6, 9) with rabbit anti CD45 antibody (A), misfolded SOD1 -3H1 (B), TDP-43 (C). Black squares show the cropping locations.

FIGURE S3 | Leukocyte derived MVs are enriched in ALS patients with a different trend from endothelial, platelet and erythrocyte derived MVs. The histogram represents the enrichment of CD45, CD31, CD61, and CD235a MVs in four representative ALS patients and CTRLs (A). Flow cytometry dot plots of MVs isolated from plasma of a representative ALS patient labelled with CD45 and CD235a (B) and CD61 and CD31(C).

mechanisms. Proc. Natl. Acad. Sci. U.S.A. 111, 3620–3625. doi: 10.1073/pnas.<PHONE_NUMBER>

**Conflict of Interest Statement:** LL is an employee of Becton Dickinson Italia S.p.A, which provided the antibodies used in this study.

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

Copyright © 2019 Sproviero, La Salvia, Colombo, Zucca, Pansarasa, Diamanti, Costa, Lova, Giannini, Gagliardi, Lauranzano, Matteoli, Ceroni, Malaspina and Cereda. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Co-cultures of Glioma Stem Cells and Primary Neurons, Astrocytes, Microglia, and Endothelial Cells for Investigation of Intercellular Communication in the Brain

Zhiyun Wei, Shubham Kale, Rachid El Fatimy, Rosalia Rabinovsky and Anna M. Krichevsky\*

Department of Neurology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, United States

#### Edited by:

Francesc Xavier Guix, Severo Ochoa Molecular Biology Center (CSIC-UAM), Spain

#### Reviewed by:

Yu Fujita, The Jikei University School of Medicine, Japan Rong Xu, La Trobe University, Australia

\*Correspondence: Anna M. Krichevsky<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Neurodegeneration, a section of the journal Frontiers in Neuroscience

Received: 17 December 2018 Accepted: 29 March 2019 Published: 18 April 2019

#### Citation:

Wei Z, Kale S, El Fatimy R, Rabinovsky R and Krichevsky AM (2019) Co-cultures of Glioma Stem Cells and Primary Neurons, Astrocytes, Microglia, and Endothelial Cells for Investigation of Intercellular Communication in the Brain. Front. Neurosci. 13:361. doi: 10.3389/fnins.2019.00361 Intercellular communication within complex biological and pathological systems via extracellular vesicles (EVs) and secreted factors is a highly attractive area of research. However, cell models enabling investigation of such communication in vitro are limited. Commonly utilized is the supplementation of hyper-concentrated EVs or other extracellular factors to the recipient cell cultures. This approach requires purification of the secreted complexes and is confounded by the contamination of media components. Two-chamber co-cultures of donor and recipient cells separated by a pore membrane may represent a more physiological and better-controlled system for the investigation of intercellular communication. Yet, distinct culture conditions for different neural cell types often make them incompatible for co-culturing. Here we optimized short-term cocultures of patient-derived low-passage glioma-initiating stem cells with normal cells of the brain microenvironment, such as primary neurons, astrocytes, microglia, and brain endothelial cells. We demonstrate the culture compatibility of these cell types and internalization of glioma-derived extracellular RNA by the normal recipient cells. The presented protocols are valuable for the investigation of intercellular communication between glioma brain tumor and cells of its microenvironment, including but not limited to the EVs-mediated communication.

#### RESEARCH IN CONTEXT

Cell-to-cell communication is essential in normal physiology and implicated in disease; however, experimental systems for its modeling in vitro are limited. Particularly, the investigation of communication between brain tumors and normal cells of the brain microenvironment has been challenged by the lack of adequate culture models. Here we developed co-cultures of glioma stem cells with various types of normal brain cells, including primary neurons, astrocytes, microglia, and brain endothelial cells, and demonstrated their utility for the study of intercellular communication. Detection of proposed markers in the recipient cells confirmed RNA transfer in these co-cultures.

Keywords: intercellular communication, extracellular vesicles, glioma stem cells, neurons, microglia, astrocytes

## HIGHLIGHTS

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## INTRODUCTION

Intercellular communication is critical for all biological and pathological processes. Established secreted mediators of such communication include hormones, neurotransmitters, growth factors, cytokines, chemokines, and neurotrophins. More recently, additional routes of transmission, such as those mediated by extracellular vesicles (EVs) (Valadi et al., 2007; Skog et al., 2008) and extravesicular ribonucleoproteins (RNPs) (Vickers et al., 2011) carrying RNA, proteins, lipids and other cargo molecules, have also been extensively explored.

Investigation of these modes of communication is an especially active area of research in the field of cancer biology, as they are thought to contribute to the invasion, metastasis, angiogenesis, chemo-resistance, and immune tolerance of many malignancies (Hanahan and Weinberg, 2011). Among these malignancies is glioblastoma (GBM), the most common and invasive primary brain tumor that affects both adult and pediatric patients. The median survival of GBM patients is only about 14–15 months, despite aggressive treatments that include tumor resection followed by radiotherapy and chemotherapy. Investigation of the tumor cross-talk with other cells of the brain microenvironment may provide new avenues for the development of targeted therapeutic applications (reviewed by Broekman et al., 2018).

The most common approaches for the investigation of the intercellular communication rely on either a single-pulse or repeated supplementation of the purified and concentrated "agent" (e.g., EVs) to the recipient cells. However, such a strategy does not consider the local physiological concentration and the dynamics of release and uptake of the investigated factors. Another way of studying intercellular communication employs co-cultures of donor and recipient cells in common culture conditions, with a membrane separating the two cell types. Comparing to the previous strategy, co-culture experiments may better model the complex and persistent communication between the cells, physiological release-uptake dynamics, and enable assessment of multiple mediators of communication between the donor and recipient cultures. Such experimental design has been utilized in many studies, including those reporting miRNA transfer from cardiac fibroblast to cardiomyocytes (Bang et al., 2014) and between dendritic cells (Alexander et al., 2105), ncRNA transfer from cardiospherederived cells to macrophages (Cambier et al., 2107), mRNA transfer from acute myelogenous leukemia to stromal niche (Huan et al., 2103), DNA transfer between parasites (Regev-Rudzki et al., 2013), protein transfer from mesenchymal stromal cells to haematopoietic cells (Bauer et al., 2011), and virus transfer from infected hepatoma cells to plasmacytoid dendritic cells (Feng et al., 2014). In these cases, both donor and recipient cells share the same, or similar, routine culturing condition and require no additional optimization for co-culturing. However, the studies of intercellular communication in the brain and brain tumors are largely hampered by the difficulty in coculturing glioma with primary neuroglial cells, as they have distinct culture conditions.

Here we screened multiple conditions and developed novel protocols for co-culturing patient-derived glioma-initiating stem cells (GSC), a key population of GBM cells that is considered most resistant to chemo- and radiotherapies and largely responsible for tumor recurrence, with primary neuronal, glial, and endothelial cells of the brain. We provide two new species-specific ncRNA markers to demonstrate extracellular RNA (exRNA) transfer from GSC to the normal cells. The established cell systems may provide a simple and physiologically relevant model for studying the bidirectional communication in malignant gliomas.

## MATERIALS AND METHODS

#### Human GSC Cultures

Low passage GBM8 cells have been cultured as neurospheres in Neurobasal medium (Gibco) supplemented with 3 mM GlutaMAX (Gibco), 1× B-27 supplement (an optimized serum-free proprietary supplement commercially available from Gibco), 0.5× N-2 supplement (a chemically defined, serum-free supplement from Gibco; components include transferrin, insulin, progesterone, putrescine and selenite), 0.5% Antibiotic-Antimycotic Solution (Corning), 20 ng/mL EGF (R&D Systems, MN, United States), and 20 ng/mL FGF (PeproTech, NJ, United States). Mature neurospheres were dissociated using NeuroCult Chemical Dissociation Kit (Mouse) (Stemcell Technologies, Canada). Approximately 0.5 × 106 cells were seeded in 10 mL fresh media in a 10 cm dish (Corning), and 1/3 volume of fresh medium was added every 3 days. Mature neurospheres were usually formed within 7∼10 days.

#### Primary Neuron Cultures

Brain cortical tissues of E18 C57BL/6 mice were dissected, dissociated with papain (12 U/ml; Worthington) for 20 min at 37◦C, and triturated to single cells using Pasteur pipettes. After dissociation, the cells were washed with DMEM-F12 (Corning) three times and pelleted at 300 g centrifugation. The cells were seeded on a poly-D-lysine (Sigma-Aldrich, MO, United States) coated 24-well plates, at 80,000 cells per cm2 . The seeding medium consisted of Neurobasal, 1× B-27, 1% Antibiotic-Antimycotic Solution (Corning), 0.5 mM GlutaMAX, and 2% FBS (Gibco). Next day, the seeding medium was replaced to the fresh culture medium with no FBS. After 5 days, the culture medium was supplemented with Ara-C (5 µM; Sigma-Aldrich) to deplete glial cells. Half of the medium was replaced with the fresh culture medium twice a week.

#### Primary Glia Cultures

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Brain cortical tissues of P1 C57BL/6 mice were cut to small pieces and dissociated with 0.25% Trypsin (Gibco) and 0.1 mg/mL DNase I (Roche) for 15 min at 37◦C, with swirling every 3 min. After dissociation, the cells were washed with DMEM-F12 three times and pelleted at 300 g centrifugation. The cells collected from three pups were seeded in one T75 flask with culture medium consisted of DMEM-F12, 10% FBS, and 1% Antibiotic-Antimycotic Solution. The medium was changed to the fresh culture medium 3 days after seeding, and further replaced every 5–7 days. For astrocyte cultures, the flasks were shaken at 200 rpm at 37◦C overnight three times to remove microglia, and then trypsinized and transferred to 24-well plates. For microglia cultures, the media was supplemented with a recombinant M-CSF mouse protein (10 ng/mL; Gibco). One week later, floating microglia were collected by pelleting the conditioned media at 300 g for 10 min, and then seeded on the poly-Dlysine coated 24-well plates, at 100,000 cells per cm2 with fresh culture media.

### Cultures of Brain Endothelial Cells

Mouse primary brain microvascular endothelial cells (MBEC) were purchased from Cell Biologics, IL, United States (Catalog# C57-6023; Lot# 070613T2MP) and cultured according to the manual, but with no heparin (as it interferes with EV uptake Atai et al., 2013; Christianson et al., 2013). Briefly, T25 flasks were pre-coated with Gelatin-based coating solution (Cell Biologics), 106 cells seeded in the Endothelial Cell Medium (Cell Biologics) and passaged 1:2 upon confluence. Low passages (1–4) have been used in this study.

## Co-culture Conditions

The recipient normal cells of the brain were seeded in 24 well plates, as described above. One day later, small GBM8 neurospheres grown in GSC conditions were transferred to the upper chamber of the Millicell hanging insert with 1.0 µm pore size (Millipore). For co-cultures of GSCs with neurons, the optimized media consisted of Neurobasal, 1× B-27, 0.5× N-2 supplement, 0.5 mM GlutaMAX, and 1% Antibiotic-Antimycotic Solution. For co-cultures of GSCs with glia, the optimized media consisted of DMEM-F12, 1× B-27, 0.5× N-2 supplement, and 1% Antibiotic-Antimycotic Solution. For co-cultures of GSCs with endothelial cells, the optimized media consisted of Mouse Endothelial Cell Basal Medium (Cell Biologics), 1× B-27, 0.5× N-2, 0.1% VEGF, 0.1% ECGS, 0.1% EGF, 0.1% Hydrocortisone, 2 mM L-Glutamine and 1% Antibiotic-Antimycotic Solution.

## Cell Imaging

Using the IN Cell Analyzer 2200 (GE Healthcare Life Sciences, PA, United States) the 24-well plates were automatically scanned with 81 photographs per well taken at days 3 and 6 of cell culturing in the experimental media. The representative images of neurospheres were adjusted by autocontrast, and for adherent monolayer cultures, additionally, by Scott 5 HDR preset of the Photoshop (Adobe, CA, United States). All images have been processed in parallel, using identical settings.

## RNA Isolation and qRT-PCR

Total RNA was isolated by miRCURY RNA Isolation Kit – Cell & Plant (Exiqon, Denmark), with on-column DNase treatment (Qiagen, Germany), after 3 days of co-culture. RNA quality was examined using Agilent 2100 Bioanalyzer (Agilent, CA, United States). qRT-PCR reactions were performed using miRCURY system (Exiqon) as previously reported (Wei et al., 2017) for small RNAs, and iScript system (Bio-Rad, CA, United States) for mRNAs, with the following DNA primers. Human RNY5 RNA (Christov et al., 2006): 50 -AGTTGGTCCGA GTGTTGTGG-30 and 50 -AACAGCAAGCTAGTCAAGCG-30 ; human RNU2-1 RNA: 50 -TTTGGAGCAGGGAGATG-30 and 5 0 -CACCGTTCCTGGAGGTA-30 ; mouse Actb: 50 -GGCACCAC ACCTTCTACAATG-30 and 50 -GGTACGACCAGAGGCATA CA-30 ; mouse Gapdh: 50 -GAAGGTCGGTGTGAACGGATT-30 and 50 -CGTGAGTGGAGTCATACTGGAAC-30 ; mouse NeuN: 5 0 -GCACGGCATGACCCTCTACA-30 and 50 -GTGGAGTTGC TGGTTGTCTGTC-30 ; mouse Tuj1: 50 -CAGCGATGAGCACGG CATAGA-30 and 50 -CCAGGTTCCAAGTCCACCAGAAT-30 ; mouse Gfap: 50 -AACGACTATCGCCGCCAACTG-30 and 50 -CG AGCAAGTGCCTCCTGGTAAC-30 ; mouse Eaat1: 50 -TGCCT CTCCTCTACTTCCTG-30 and 50 -CCACACCATTGTTCTCTT CCA-30 ; mouse CD11b: 50 -AGCACCTCGGTATCAGCATAT TG-30 and 50 -GGTATTGCCATCAGCGTCCAT-30 ; mouse Iba1: 5 0 -CAGACTGCCAGCCTAAGACA-30 and 50 -GGATCATCGA GGAATTGCTTGT-30 ; mouse CD31: 50 -TCCAACAGAGCCAG CAGTATGA-30 and 50 -GCGATGACCACTCCAATGACAA-30 ; mouse Cdh5: 50 -CCAGCGACACTTCTACCACTTC-30 and 5 0 -CTGTCACTGGTCTTGCGGATG-30 . The U6 snRNA was quantified with LNA-containing primers (Exiqon) and used as the endogenous control for small RNAs.

#### Western Blot

Cell lysates containing equal amount of total proteins were separated by SDS-PAGE with 4–12% Blot Bis-Tris Plus Gels (Thermo Fisher Scientific), and transferred to 0.45 µm PVDF membrane (Thermo Fisher Scientific). After blocking with 5% (wt/vol) fat-free milk in Tris-buffered saline with 0.075% Tween-20 (TBST), the membranes were incubated overnight with diluted primary antibodies (Tuj-1: Abcam ab14545, 1:10000 dilution; GFAP: Sigma-Aldrich G9269, 1:7500 dilution; CD11b: Abcam ab133357, 1:2000 dilution; CD31: Cell Signaling Technology 3528S, 1:500 dilution; β-Actin: Abcam ab3280, 1:10000 dilution) at 4◦C. The membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology 7074S and 7076S, 1:5000 dilution) for 1 h at room temperature. The blots were developed by the Amersham ECL Reagent (GE Healthcare).

## RESULTS

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## GSCs Cultured in Alternative Conditions Change Their Morphology

For maintenance of the stem-cell-like properties, GSCs are typically cultured in a defined serum-free Neurobasal media, whereas primary astrocytes, microglia, and endothelial cells are cultured in distinct growth conditions, supplemented with FBS. Optimal growth conditions for GSCs and primary neurons are more similar, with the supplementation of N-2 and additional growth factors in GSC media, which suggest their better compatibility in the co-cultures.

To investigate whether GSCs can maintain their morphology in conditions used for culturing normal cells of the brain, we established small spheroids of GBM8 GSCs, and at day 3 after passaging, replaced the GSC growth medium with the media corresponding to either neuronal, glial, or endothelial cells, followed by the observation of GSC morphology over 6 days. As shown in **Figure 1A**, when cultured in the regular GSC medium, in 9 days GSCs grow into spheroids of variable sizes. Similar,

medium (L) and their morphological changes in GSC medium (K; observed in 78 out of 81 images). Typical morphological alterations are depicted by arrows,

arrowhead, and dashed lines. Images were taken 6 days after medium change. N = at least two independent experiments/condition.

albeit smaller GSC spheroids are formed in the neuronal media lacking N-2 supplements (**Figure 1B**). However, as expected, when cultured in the FBS-containing media used for glial or endothelial cells, small gliomaspheres became adherent to the plate surface and exhibit altered morphology, indicative of their differentiation (**Figures 1C,D**). Most of the cells spread out of the initial spheres to form monolayer or multilayer colonies. In the glial medium that is essentially similar to the media used for GSC differentiation, GSCs developed multiple extended processes (arrows in **Figure 1C**) with frequent branching (arrowhead in **Figure 1C**), resembling the premature primary astrocytes (observed in 81 out of 81 images analyzed). In the endothelial medium, GSCs exhibit fibroblastic morphology with bipolar or multipolar elongated shapes (arrows in **Figure 1D**; observed in 81 out of 81 images analyzed).

### Primary Brain Cells Change Their Morphology When Cultured in GSC Media

To examine the compatibility of primary neurons, astrocytes, microglia, and endothelial cells with GSC medium, we established individual cultures of these cells using their optimal growth conditions, then replaced the media with the GSC medium, and observed their growth over 6 days. Primary neuronal cultures grown in the GSC media at DIV10 to 16 exhibited altered cell morphology and reduced complexity, with fewer and less branched processes (arrows in **Figure 1E**, compared to **Figure 1F**).

Primary astrocytes usually maintain their morphology and remain adherent and healthy for several months, as shown in **Figure 1H**. However, when the pre-established monolayer of astrocytes was exposed to the GSC medium, the cells dramatically changed their shape, become slender and more elongated, and the cultures lost their typical dense monolayer appearance (arrows in **Figure 1G**).

Microglia cultured in the established optimal conditions is firmly attached to the polylysine matrix, displays a typical ramified morphology, and remains quiescent (**Figure 1J**). However, when cultured in the GSC media, microglia became activated and proliferative, giving rise to large colonies (area surrounded by dashed lines in **Figure 1I**).

Finally, the cultures of primary brain endothelial cells (**Figure 1L**) exhibited significant cell death and quickly degenerated when grown in the GSC medium (**Figure 1K**). Taken together, these results indicate that the culture conditions utilized for GSCs and normal primary cells of the brain are incompatible and cannot be for co-culture experiments.

#### Optimization of Co-culture Conditions for GSC and Primary Normal Cells of the Brain

To optimize the GSC co-cultures with normal cells of the brain, for each pair of cell types we tested several media compositions based on their original recipes (**Supplementary Table S1**) and observed the cell growth and morphology over time. The co-culture conditions were optimized to enable the maintenance of the undifferentiated neurosphere state by GSCs (**Figures 2A–C**) and preserve the original morphologies of the counterpart normal cells (**Figures 2D–G**) for at least 6 days. Loss of neuronal processes has been rarely observed in the optimized conditions (in only 4/81 images in the co-culture conditions, which is similar to 3/81 rate observed in normal neuronal cultures). Abnormal morphologies were not observed for astrocytes, microglia or endothelial cells in the optimized co-culture conditions. As expected, the B27 supplement was necessary to maintain the undifferentiated status of GSCs while FBS triggered their differentiation. Growth factors (EGF and FGF) appear to significantly alter the morphology of neurons and astrocytes and induce proliferation of microglia. They have been, therefore, omitted from the corresponding co-culture media. On the other hand, replacing FBS with B27 did not trigger significant morphological changes of primary glial and endothelial cells. Glutamine supplementation had significant effects on GSC and neurons (with low concentrations being toxic to the former and high concentrations to the latter) and, thus, had to be balanced for their co-cultures. N2 supplementation did not have major effects on the co-cultures. Furthermore, mRNA and protein levels of the representative markers for each recipient cell type have not changed under the corresponding co-culture conditions (**Figure 3**). Final compositions of the media supporting the co-cultures of GSCs with normal neurons, astrocytes, microglia, and brain endothelial cells are described in the Section "Materials and Methods."

#### Validation of Intercellular RNA Transfer in Co-cultures

To validate a cross-talk between GSC and normal cells in the optimized co-culture conditions, human GSCs were co-cultured with various types of primary mouse brain cells, including neurons, astrocytes, microglia and endothelial cells, and the transfer of GSC-derived exRNA to the normal cells was assessed. The RNA was isolated from the recipient normal mouse cells after 3 days of co-culturing, and the levels of specific RNA markers in these cells were examined. We tested the transfer of snRNA and Y RNA, two classes of ncRNA abundantly released by GSCs (Wei et al., 2017). While human-specific RNAs such as RNU2-1 snRNA and RNY5 were undetectable in the monocultures of mouse primary microglia, they were strongly elevated in the microglia co-cultured with human GBM8 neurospheres (**Figure 4**). Similar results were observed for the recipient primary cultures of neurons, astrocytes, and endothelial cells (**Figure 4**). These experiments confirmed RNA transfer from human GSCs to the mouse recipient cells in co-cultures and thus demonstrated the application of this protocols for investigation of the intercellular communication between GSCs and normal cells of glioma microenvironment in vitro.

## DISCUSSION

Although it is gaining increased attention in recent years, intercellular communication in the brain tumor microenvironment remains among the least investigated

FIGURE 2 | GSC and normal cell types of the brain maintained in the optimized co-culture conditions for 6 days. (A–C) GBM8 spheroids in co-culture media optimized for neurons (A), glia (B), and endothelial cells (C).  N = at least two independent experiments/condition.

topics in the pathology of malignant gliomas. The impact of the intercellular interactions occurring in the brain on the glioma initiation, growth, and therapy resistance has not been thoroughly elucidated. Previous studies mostly focused on the secreted molecules that mediate communication, such as hormones, growth factors, cytokines, and chemokines. EVs and extravesicular RNP complexes carrying heterogeneous cargo (e.g., proteins, nucleic acids, and lipids) have been proposed as additional potent and more complex communication vehicles. Notably, the in vitro modeling of such complex interactions underlying the growth of intracranial tumors remains one of the major impediments in the field.

Most studies of the intercellular communication in vitro rely on the pulse supplementation of an experimental factor derived from the donor cells to the recipient cells. While such traditional strategy is merited for specific molecular agents/mediators (e.g., cytokines), it can be suboptimal for more complex mediators, such as EVs. It is currently unclear how to dose and time this type of experiments so that they mimic the physiology and dynamics of EV/RNP release and uptake; therefore, it is common to simply apply a pulse of hyper-concentrated preparations of EVs to the recipient cells. In addition, since any fresh media contain active ingredients that may cause falsepositive results, designing suitable controls for such experiments is challenging (Wei et al., 2016; Tosar et al., 2017). Co-culturing diverse cell types may provide the better, more physiological approach for studying the intercellular communication among them. However, the caveat, especially concerning the difficultto-culture primary and low passage cells of the brain and brain tumors, is their poor compatibility in culture. The cells of the CNS such as neurons, astrocytes, microglia, and brain derived endothelial cells are highly sensitive and require specific, distinct conditions for their growth in vitro. Optimized protocols for coculturing these cells with glioma will advance the investigation of intercellular communication between brain tumors and brain microenvironment, mediated by EVs and other means (Broekman et al., 2018).

Here we optimized conditions for co-culturing four major types of normal brain cells with glioma-initiating cells so that they could be maintained together and retain their normal phenotypes for at least 6 days. This time frame is generally sufficient for a diverse set of molecular and phenotypic assays. In addition to revealing the impact of a broad EV population or the entire secretome, specific EV subclasses varying by their sizes (e.g., oncosomes, microvesicles, and exosomes) can be studied in such two-chamber systems separated by the membranes with different pore sizes. For example, modification of the assay to employ the inserts with either 0.2 µm and 20 nm pores will assess the relative contributions of EVs and RNPs.

In this study, we also propose several human-specific RNA markers that provide positive controls for RNA transfer studies and can be utilized for the studies of exRNA secretion and uptake mechanism. While the focus of exRNA field remains largely on miRNAs due to their enrichment in EVs and important regulatory potential, other classes of regulatory transcripts are abundantly released by various types of cells (Wei et al., 2017; Chiou et al., 2018; Driedonks and Nolte-'t Hoen, 2018; Hinger et al., 2018; Rimer et al., 2108; Elsemuller et al., 2019). The copy numbers of some non-miRNA transcripts in the extracellular space are significantly higher than those of even the most abundant miRNAs (Wei et al., 2017). While the biological impact of their intercellular transfer is largely unknown, the presented protocols may aid the corresponding investigation. Here we demonstrated the transfer of two classes of highly abundant ncRNA, snRNA, and Y RNA, from glioma cells to the normal cells of the brain. Overall, the established protocols are valuable for the investigation of intercellular communication between glioma brain tumor and its microenvironment, including but not limited to the EVsmediated communication.

#### ETHICS STATEMENT

All animal experiments have been approved by the Brigham and Women's Hospital Institutional Animal Care and Use Committee.

## AUTHOR CONTRIBUTIONS

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AK conceived the study. ZW and AK designed the experiments and wrote the manuscript. ZW, SK, and REF performed the experiments. RR assisted with experiments. All authors revised and approved the manuscript.

## FUNDING

This work was supported by the U19 CA179563 grant and U19 administrative supplement through the NIH Common Fund and the Office of Strategic Coordination/Office of the NIH Director, R01 CA215072, and R21 NS098051 grants.

#### REFERENCES

#### ACKNOWLEDGMENTS

We thank Ramil Arora for assisting with experiments and members of Krichevsky laboratory for helpful discussions.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fnins. 2019.00361/full#supplementary-material

TABLE S1 | Tested medium compositions and their effects on cell growth and morphology. Optimal medium compositions for co-cultures are highlighted in red. N = at least two independent experiments/condition.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Wei, Kale, El Fatimy, Rabinovsky and Krichevsky. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Neuron-Derived Exosome Proteins May Contribute to Progression From Repetitive Mild Traumatic Brain Injuries to Chronic Traumatic Encephalopathy

#### Edward J. Goetzl1 \*, Aurélie Ledreux2 \*, Ann-Charlotte Granholm2 , Fanny M. Elahi3 , Laura Goetzl4 , Jade Hiramoto5 and Dimitrios Kapogiannis6 \*

#### Edited by:

Francesc Xavier Guix, Severo Ochoa Molecular Biology Center (CSIC-UAM), Spain

#### Reviewed by:

Ye Xiong, Henry Ford Health System, United States Bogdan A. Stoica, University of Maryland, Baltimore, United States

#### \*Correspondence:

Edward J. Goetzl<EMAIL_ADDRESS>Aurélie Ledreux<EMAIL_ADDRESS>Dimitrios Kapogiannis<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Neurodegeneration, a section of the journal Frontiers in Neuroscience

Received: 22 February 2019 Accepted: 23 April 2019 Published: 08 May 2019

#### Citation:

Goetzl EJ, Ledreux A, Granholm A-C, Elahi FM, Goetzl L, Hiramoto J and Kapogiannis D (2019) Neuron-Derived Exosome Proteins May Contribute to Progression From Repetitive Mild Traumatic Brain Injuries to Chronic Traumatic Encephalopathy. Front. Neurosci. 13:452. doi: 10.3389/fnins.2019.00452 1 Department of Medicine, University of California, San Francisco, San Francisco, CA, United States, 2 Knoebel Institute for Healthy Aging, University of Denver, Denver, CO, United States, 3 Department of Neurology, Memory and Aging Center, University of California, San Francisco, San Francisco, CA, United States, 4 Department of Obstetrics, Gynecology and Reproductive Sciences, Health Sciences Center at Houston, University of Texas, Houston, TX, United States, 5 Division of Vascular Surgery, Department of Surgery, University of California, San Francisco, San Francisco, CA, United States, 6 Laboratory of Neurosciences, Intramural Research Program, National Institute on Aging, Baltimore, MD, United States

The recent recognition that Alzheimer disease-like pathology may be found in chronic traumatic encephalopathy (CTE) even after acute mild traumatic brain injury (mTBI) has increased the urgency of elucidating mechanisms, identifying biomarkers predictive of high risk of development of CTE, and establishing biomarker profiles indicative of impactful effects of treatments. Of the many proteins that are loaded into neuron-derived exosomes (NDEs) from damaged neurons after acute TBI, the levels of prion cellular protein (PRPc), coagulation factor XIII (XIIIa), synaptogyrin-3, IL-6, and aquaporins remain elevated for months. Prolonged heightened expression of aquaporins and IL-6 may account for the persistent central nervous system edema and inflammation of CTE. PRPc, XIIIa and synaptogyrin-3 bind and concentrate neurotoxic forms of oligomeric amyloid β peptides or P-tau for delivery into neurons at or distant from the site of trauma. Our progression factor hypothesis of CTE asserts that physiological neuronal proteins, such as PRPc, XIIIa, synaptogyrin-3, IL-6 and aquaporins, that increase in concentration in neurons and NDEs for months after acute TBI, are etiological contributors to CTE by either direct actions or by recruiting neurotoxic forms of Aβ peptides or P-tau. Such progression factors also may be useful new targets for development of drugs to prevent CTE.

#### Keywords: amyloid, cellular prion protein, phospho-tau, synaptogyrin-3, Alzheimer disease

**Abbreviations:** Aβ42, β amyloid peptide 1-42; APP, amyloid precursor protein; BACE-1, beta-secretase-1 or beta-site amyloid precursor protein cleaving enzyme 1; BBB, blood-brain barrier; CTE, chronic traumatic encephalopathy; DU, University of Denver; MCP, monocyte chemoattractant protein; mTBI, mild TBI; NDE, neuron-derived exosome; NKCC-1, sodium-potassium-chloride co-transporter-1; PRPc, cellular prion protein; Rab 7, Rab 10, Rab 35, ras-related small GTPase 7, 10, 35; TBI, traumatic brain injury; UC, University of California; UCH-L1, ubiquitin C-terminal hydrolase L1; XIIIa, coagulation factor XIII.

## INTRODUCTION

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Traumatic brain injury (TBI) is the term used to describe mechanical injury to the parenchymal tissues and meninges of the brain, and the subsequent series of further damaging proteolytic, oxidative, inflammatory, and proteinopathic responses (Langlois et al., 2006; Roozenbeek et al., 2013; Quillinan et al., 2016). Several million TBIs occur annually in the United States, of which approximately 80% are defined as mild traumatic brain injury (mTBI) (Faul and Coronado, 2015; Taylor et al., 2017). A high frequency of repetitive mTBIs is observed in young adults from participation in sports and from the shock waves created by military explosives (Broglio et al., 2009; Champion et al., 2009; Wilk et al., 2012; Papa et al., 2015; McKee et al., 2016). It is clear that moderate and severe TBI predispose to both more prevalent and earlier onset Alzheimer disease, and to CTE (Barnes et al., 2014; Gardner et al., 2014; McKee et al., 2016; Perry et al., 2016). Despite the far higher prevalence of mTBI than moderate and severe TBI, it has only recently been recognized that repetitive mTBIs also increase the risk of subsequent development of CTE and dementia (Crane et al., 2016; Barnes et al., 2018). Our understanding of the mechanisms of acute mTBI resulting from a single concussive blow and CTE following repeated episodes of acute mTBI is incomplete, in part due to the past lack of sensitive and accurate biomarkers for the many constituent abnormalities.

The complex matrix of pathways interacting in the course of an mTBI begin with disruption of the blood-brain barrier (BBB) and axonal injury. BBB damage is manifested maximally within hours by morphological changes in endothelial cells, pericytes and astrocytes, increases in the number of perivascular monocytes, and elevated blood levels of tight-junction and astrocyte-specific proteins (Blyth et al., 2009; Higashida et al., 2011; Obermeier et al., 2013; Kuriakose et al., 2018). Subsequent edema of neurons and interstitial tissues follows with increased brain tissue levels of proteins from damaged neuronal framework structures, channels, transporters, and vesicle transport systems (Hue et al., 2016; Kuriakose et al., 2018). Inflammation develops in the same time period of initial responses to acute mTBI, as shown by increased brain tissue levels of cytokines, such as IFN-α, IL-1β, IL-6, and MCP, oxygen radicals and matrix metalloproteinases (Helmy et al., 2011; Woodcock and Morganti-Kossmann, 2013; Yan et al., 2014; Gyoneva and Ransohoff, 2015; Lozano et al., 2015; Simon et al., 2017; Roselli et al., 2018). This period also is characterized by increased expression of stress response and pro-apoptotic factor genes (Mettang et al., 2018).

Onset of more persistent features of CTE months or years after a TBI may be foreshadowed by sustained neuronal elevations of elements of proteinopathic neurodegeneration and chronic inflammation (DeKosky et al., 2013). Although neuropathogenic bridges linking acute mTBI to CTE are poorly understood, some mechanisms have been recognized. Within 24 h of acute TBI in rats, hippocampal and cortical brain tissues showed transient increases in beta-secretase-1 (BACE-1) (Blasko et al., 2004). In humans suffering single-incident severe TBI, increases in brain tissue levels of APP and Aβ peptides as well as diffuse Aβ plaques may be found within hours and persist variably (DeKosky et al., 2013). In contrast, the predominant early neuropathology in CTE following repetitive mTBIs is patchy distribution of neuropil threads and neurofibrillary tangles of P-tau throughout the neocortex (DeKosky et al., 2013). Plasma levels of tau-positive exosomes were abnormally elevated in former professional football players, exposed to repetitive mTBIs, and these increases correlated with decreases in memory and psychomotor speed (Stern et al., 2016). Although many abnormalities of cognition and affect or mood are similar in subjects suffering from CTE after acute severe TBI or repetitive mTBI, relationships between these neurocognitive abnormalities and differing proteinopathic etiologic mechanisms have not been delineated (Prince and Bruhns, 2017; Roy et al., 2017; Dailey et al., 2018).

Investigations of a wide range of neurally derived proteins and of systemic predominantly lipid metabolites in CSF and plasma have not yielded biomarkers that reliably assess the severity of mTBI, predict progression of mTBI to CTE, or offer useful targets for therapy (DeKosky et al., 2013; Agoston et al., 2017; Werhane et al., 2017; Kim et al., 2018). Changes in blood levels of some individual proteins, such as tau, s100B, neurofilament light chain, ubiquitin protein hydrolase-1 and neuron-specific enolase, showed moderate diagnostic and/or prognostic value in some circumstances, but had limited neural specificity, late expression and lack of cross-correlation (Di Battista et al., 2013). A comprehensive gas chromatographic analysis of sera obtained within 2 weeks of mTBI or more severe TBI showed elevations of two medium-chain fatty acids and 2,3-bisphosphoglyceric acid, that were significantly associated with the severity of TBI and added value to clinical and radiological imaging results (Oresic et al., 2016). Six plasma metabolites, including two fatty acids, three phospholipids and tauroursodeoxycholic acid, that were quantified at <6 h to 7 days after mTBI, significantly distinguished mTBI athletes from matched uninjured teammate controls throughout the week after mTBI (Fiandaca et al., 2018). However, as for the prior investigation of metabolomic lipids, these analytes have no known specific CNS functions and statistical characteristics of data for two independent cohorts showed major differences. It thus seems appropriate now to investigate subjects with acute or chronic repetitive mTBI by quantifying alterations in levels of their endosomal and other membrane CNS proteins, that are accessible in the protected cargo of plasma NDEs. The multiphasic and multicomponent set of events characteristic of all TBIs has suggested the importance of selecting biomarkers that probe both intracellular processes and diverse relevant mechanisms.

## PLASMA NEURON-DERIVED EXOSOME PROTEIN ABNORMALITIES IN mTBI

Application of established technology to enrich the NDE subset of extracellular vesicles in plasma has permitted initial elucidation of the changes in exosome trafficking and levels of cargo proteins characteristic of mTBIs. A study of military personnel suffering mTBI and subsequent chronic unspecific CNS symptoms revealed significant increases in CD81 exosome marker-normalized NDE levels of total tau, Aβ42, and IL-10 relative to deployed controls without a history of mTBI

(Gill et al., 2018). Although the precise timing of episodes of mTBI was not determinable, most had been in combat at least once during the year preceding testing. Significant associations were identified between post-concussive symptoms and increases in NDE tau and between symptoms of post-traumatic stress disorder and increases in NDE IL-10 (Gill et al., 2018).

A recent investigation of plasma levels of NDEs and a broader profile of NDE cargo proteins has been conducted in college athletes aged 18–26 years, who provided plasma within 1 week after a sports-related mTBI (acute mTBI, n = 18) or 3 months or longer after two to four mTBIs (chronic mTBI, n = 14) (Goetzl et al., 2019). Plasma levels of NDEs, assessed by closely correlated counts and levels of extracted CD81 exosome marker, were significantly depressed by a mean of 45% in acute mTBI (P < 0.0001), as compared to teammates without TBI (n = 21), but not in chronic mTBI. Mean CD81-normalized NDE concentrations of a range of functional brain proteins were significantly abnormal relative to those of uninjured teammate controls in acute but not chronic mTBI groups, including ras-related small GTPase 10 (Rab 10), 73% decrease; annexin VII, 8.8-fold increase; UCH-L1, 2.5-fold increase; αII spectrin fragments, 1.9-fold increase; claudin-5, 2.7-fold increase; and sodium-potassium-chloride cotransporter-1 (NKCC-1), 2.8-fold increase (all acute increases were significantly different from controls, P < 0.0001). Three physiological neural proteins whose NDE levels changed significantly in acute mTBI also maintained lesser alterations in chronic TBI. Aquaporin 4 showed 8.9-fold (P < 0.0001) and 3.6-fold (P = 0.0004) increases, and synaptogyrin-3 showed 3.1-fold (P < 0.0001) and 1.3-fold (P = 0.041) increases, whereas ras-related small GTPase 35 (Rab 35) exhibited 56% (P < 0.0001) and 33% (P = 0.0045) decreases, respectively, in acute and chronic mTBI. In chronic mTBI, there were elevated CD81-normalized NDE levels of usually neuropathologic β-amyloid peptide 1–42 (Aβ42), 1.6-fold (P < 0.0001 relative to controls); XIIIa, 1.3-fold (P = 0.042); P-T181-tau, 2.2-fold (P < 0.0001); P-S396-tau, 1.6 fold (P < 0.01); IL-6, 16-fold (P < 0.0001); and prion cellular protein (PRPc), 5.1-fold (P < 0.0001), with lesser or greater (XIIIa, IL-6, PRPc) increases in acute mTBI.

The acute decreases in Rab 10 and Rab 35 levels in NDEs, that are presumed to reflect neuronal levels of these small GTPases involved in vesicular formation, trafficking and secretion, may account for the observed acute decreases in plasma levels of NDEs, as discussed in the next section (**Figure 1**). The sustained decrease in NDE level of Rab35 distinguishes this response from the transient acute decrease in NDE level of Rab10. Levels of other NDE cargo proteins, that represent disposal of damaged neuronal proteins, instead significantly increased after acute mTBI but these increases were not all sustained in chronic mTBI. Of the acute mTBI NDE cargo responders, annexin VII has roles in neuronal survival and membrane transport functions, UCH-L1 targets damaged proteins by ubiquitination for elimination, αII spectrin with actin constitutes the neuronal periodic skeleton, claudin-5 is a component of the tight junction of the BBB, and NKCC-1 and aquaporins, respectively control ion and water movements required for normal neuronal functions (Creutz, 1992; Ohara et al., 1998; Brophy et al., 2011; Scharfman and Binder, 2013; Morita et al., 2014). In contrast, the NDE proteins that increased in concentration after chronic repetitive mTBIs without (only P-S396-tau) or with significant increases in acute mTBI are regarded predominantly as mediators of proteinopathic neurodegeneration, including total tau, P-T181-tau, P-S396-tau, Aβ42, IL-6, PRPc, and XIIIa. Of this group, PRPc and XIIIa, as well as the acute responder synaptogyrin-3 remain elevated through repetitive mTBIs and bind, concentrate and deliver the mediators of proteinopathic neurodegeneration P-tau or Aβ42 to other neurons.

Hypoxic or ischemic CNS damage in two disease states evoked some of the same changes in plasma levels of NDEs and/or cargo proteins as were observed in mTBI. In the first example, ten patients undergoing multi-branched endovascular repair of aortic aneurysms were studied pre-operatively, immediately postoperatively and the day after surgery. This form of surgery is often associated with ischemic hyperglycemic spinal damage and prolonged lower extremity weakness despite application of intraoperative counter-measures (Hiramoto et al., 2017). The mean level of NDEs fell significantly after surgery and returned the following day to that observed pre-operatively. The NDE mean level of Rab 10 also decreased post-operatively and rose to near normal the next day. NDE mean levels of PRPc, neurofilament light chain, NKCC-1, aquaporins and several synaptic proteins increased significantly after surgery and decreased to near normal the next day. This contrasts with the sustained elevations of PRPc, aquaporins and synaptogyrin-3 after mTBI. NDE levels of differently phosphorylated forms of insulin receptor signaling protein, changed post-operatively in accordance with development of insulin resistance. The mean NDE level of several other proteins, such as IL-6, rose post-operatively, and fell the next day, but remained significantly higher than that seen preoperatively, which again raised the possibility of a subset of neural protein progression factors that might mediate subacute or chronic injury after hypoxia or ischemia.

The second example is neonatal hypoxemic-ischemic encephalopathy and early identification of affected infants that are not responding to standard therapeutic hypothermia. Neonatal NDEs in plasma obtained at 8, 10 and 14 h after initiation of hypothermia were enriched by absorption with anti-Contactin-2 antibody (Goetzl et al., 2018). CD81-normalized levels of neonatal NDE synaptopodin, synaptophysin, neuronspecific enolase and mitochondrial cytochrome C oxidase were quantified immunochemically. The most promising marker for all clinical outcomes was NDE synaptopodin, as the slope of change in its NDE level between 8 and 14 h of hypothermia was associated with clinically significant outcomes including persistent seizure activity requiring treatment (p = 0.02) and length of hospital stay (P < 0.001).

#### Rab SMALL GTPases AND NDE TRAFFICKING in mTBIs

Rab small GTPases, the largest branch of the Ras superfamily, are highly abundant, and diverse organizers of intracellular membrane trafficking in the CNS (Kiral et al., 2018). The state

of membrane association and activity of Rab GTPases are highly dependent on controlling interactions with numerous classes of accessory proteins. In the CNS, most Rab GTPases have multiple functions. For example, Rab 7 in neurons is a critical regulator of multivesicular body maturation from early endosomes, fusion of multivesicular bodies with lysosomes to form autolysosomes, and retrograde axonal transport of vesicles with the motor protein dynein into neuron cell bodies. Similarly, Rab 35 is important for the rejuvenation of synaptic vesicles from sorting/recycling endosomes and influences neuronal actin dynamics, largely by activating Rho GTPases (Chua et al., 2010). In contrast to the increases in some neuronal Rab GTPases observed in many neurodegenerative diseases (Kiral et al., 2018), NDE levels of Rab 7, Rab 10, and Rab 35 decrease in acute mTBI. Any NDE-related functional significance of decreased levels of some Rab GTPases in acute mTBI remains to be established.

### POTENTIAL NDE PROTEIN PROGRESSION FACTORS IN THE CONTINUUM FROM REPETITIVE mTBIs TO CHRONIC TRAUMATIC ENCEPHALOPATHY (CTE)

It is too early in the course of elucidating acute mTBIinitiated pathways of pathogenesis of CTE for meaningful speculation about critical mechanisms. Further, we have no data for contributions of NDE miRNAs or lipids, or of astrocyteor microglia-derived exosomes. However, a tentative analytical

integration of existing data has identified several proteins in NDEs that may serve as factors mediating progression from repetitive mTBIs to CTE. Neuronal proteins that are elevated in NDEs acutely after mTBIs and remain significantly increased for 3 months or longer at levels higher than seen acutely include the directly neurotoxic Aβ42 and P-T181-tau (Goetzl et al., 2019). Neuronal proteins that are elevated in NDEs acutely after mTBIs and remain significantly increased for 3 months or longer at levels lower than seen acutely are aquaporins, synaptogyrin-3 and XIIIa, which lack direct neurotoxicity, as well as IL-6 and PRPc, that may diminish neuronal viability (Goetzl et al., 2019). Each of these latter mTBI-generated proteins is a candidate progression factor that promotes sustained neurotoxicity indirectly through enhancement of Aβ42 or P-tau effects or by other mechanisms.

In the model proposed, mTBI damages and releases from neuronal membranes and other structures a wide range of functional proteins that are loaded into NDEs in endosomal pathways (**Figure 1**). No relationships have yet been established between the severity of a single mTBI and the magnitude or duration of increases in NDE levels of the proteins being removed by the exosomal pathway, but indirect evidence suggests that most such increases last for less than 3 months after a single mTBI. Transient decreases in NDE levels and presumptively neuronal levels of Rab 7, 10, and 35 reduce formation and secretion of NDEs for days after an acute mTBI with potential back-up of released proteins in the neurons. mTBI increases NDE and neuronal levels of the binding partners PRPc with Aβ peptides, especially oligomeric forms of Aβ (Aβo), synaptogyrin-3 with P-tau species, and IL-6 with soluble IL-6 receptors (sIL-6Rs). Neurotoxic concentrations of PRPc-Aβ peptide complexes at dendrites, synaptogyrin-3- P-tau complexes at synapses, and IL-6-sIL-6R complexes accumulate both in and on neurons of origin at the site of mTBI and in NDEs that transport them into neighboring neurons not damaged directly by the inciting mTBI (**Figure 1**). Sustained increases in aquaporins promote edema and in IL-6 maintain chronic inflammation after mTBIs. Thus acute changes of mTBI may progress to pathogenesis of early CTE with increasing probability after subsequent repetitive mTBIs (**Figure 2**).

The postulated stages of mTBI induction of CTE are illustrated schematically from normal to mild deterioration after one mTBI to advanced CTE pathology after repeated mTBIs (mTBIn) (**Figure 2**). After a single mTBI, APP and BACE1 in exosomes from reactive astrocytes (AG) and to a lesser extent from damaged neurons are transferred to lesional and distant neurons. This initiates increased neuronal production of Aβ peptides, their

Microglia (MG) and astrocytes (AG) produce MDEs and ADEs, respectively, that carry increased levels of APP, BACE-1, and IL-6 capable of further neurotoxic damage of neurons that receive these other sets of exosomes. This series of processes increases with subsequent episodes of mTBI and is enhanced by other progression factors, such as XIIIa that promotes oligomerization of Aβ to Aβo. P-tau neuropil fibers and Aβo deposits increase in size and density with repetitive mTBIs and lead to neuronal apoptosis. Color depictions are green for neuronal boundary, blue for the degenerating nucleus, yellow for Aβ, P-tau deposits, and red for NDEs with progression factor complexes and AG/MG products.

oligomerization to Aβo that is catalyzed in part by increased levels of XIIIa (Hur et al., 2019), and beginning aggregation into loose deposits on neurons (**Figure 2**, second stage). IL-6 sIL-6R complexes in exosomes from reactive AG and microglia (MG) are secreted in part in their respective ADEs and MDEs for transfer to neurons, which mediates a sustained neuronal inflammatory response. PRPc-Fyn kinase-Aβ peptide complexes with XIIIa-oligomerized Aβ (Aβo) are transferred by NDEs to dendrites of other neurons and synaptogyrin-3 (SNGY-3)- P-tau complexes are transferred by NDEs and concentrated in axonal and presynaptic areas of other neurons to begin formation of loose P-tau strands and tangles. Repetitive mTBIs result in enhancement of all of these exosomal transfer processes with greater neuronal proteinopathic deposition of Aβo and forms of P-tau followed by early neuronal apoptosis in stage 3 (**Figure 2**). In summary, mTBI-derived CTE progression factors such as PRPc, XIIIa and SNGY-3, may act by binding to, concentrating and delivering to neurons neurotoxic Aβ peptides and various forms of P-tau. Others act independently of neurotoxic mediators, as exemplified by IL-6, which is bound by sIL-6Rs that dominate in neural tissues, link as the IL-6 sIL-6R complex to glycoprotein 130 on neurons and transmit neurodegenerative signals to the affected neurons (Erta et al., 2012; Rothaug et al., 2016). Accessory ligands for aquaporins have not been identified.

The many means for validation of the progression factor hypothesis of mediation of CTE by repetitive mTBIs include simple definitions of the time-courses of changes in NDE levels of three classes of relevant biomarkers. First, NDE levels of acute biomarkers, such as annexin VII, claudin-5, UCH-L1, spectrin fragments and NKCC-1, should only be elevated in the several weeks after each mTBI. Second, acute NDE increases in levels of Aβ peptide and P-tau mediators of proteinopathic toxicity should increase progressively and persist longer after each episode of mTBI. Third, NDE levels of the progression factors XIIIa, PRPc, SNGY-3, and possibly IL-6 and aquaporins should remain on elevated plateaus for months to years between episodes of mTBI. It also remains to be established if there are any relationships between the severity of a single mTBI or the stage of advancement to CTE and the NDE level of any of the biomarkers. Changes in the NDE level and duration of elevation of some biomarkers of mTBI also hopefully may provide information about the effectiveness of a therapeutic agent or program. Finally, this model may encourage extending drug discovery for mTBI and possibly Alzheimer disease to agents

#### REFERENCES

that alter neuronal expression of progression factors and their binding of Aβ peptides and P-tau species.

#### DATA AVAILABILITY

The datasets for this manuscript are not publicly available because data are available in Goetzl et al. (2019) cited reference. Requests to access the datasets should be directed to<EMAIL_ADDRESS>
### ETHICS STATEMENT

This study was carried out in accordance with the recommendations of the University of California, San Francisco, San Francisco, CA, United States and University of Denver Human Research Institutional Review Committees with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the University of California, San Francisco and University of Denver Human Research Institutional Review Committees.

## AUTHOR CONTRIBUTIONS

EG and DK formulated the conceptual framework of this perspective and prepared the draft manuscript. All authors analyzed the findings reported, edited the manuscript, and approved the final version submitted.

#### FUNDING

The research described was supported by a pilot grant to Drs. Ledreux and Granholm-Bentley from the Knoebel Institute for Healthy Aging and by support of Drs. Mustapic and Kapogiannis from the Intramural Program of the National Institute on Aging of the NIH.

#### ACKNOWLEDGMENTS

The authors are grateful to Judith H. Goetzl for preparation of the graphic illustrations.

hydrolase-L1 (UCH-L1) in severe traumatic brain injury patient biofluids. J. Neurotrauma 28, 861–870. doi: 10.1089/neu.2010. 1564

model of blast-induced traumatic brain injury. J. Neurotrauma 33, 1202–1211. doi: 10.1089/neu.2015.4067

J. Neuropsychiatry Clin. Neurosci. 29, 334–342. doi: 10.1176/appi.neuropsych. 16050088

association with postdeployment symptoms. Psychosom. Med. 74, 249–257. doi: 10.1097/PSY.0b013e318244c604

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The handling Editor declared a past co-authorship with one of the authors DK.

Copyright © 2019 Goetzl, Ledreux, Granholm, Elahi, Goetzl, Hiramoto and Kapogiannis. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Propagation of Tau via Extracellular Vesicles

#### Mar Pérez1 , Jesús Avila2,3 and Félix Hernández2,3 \*

1 Departamento de Anatomía Histología y Neurociencia, Facultad de Medicina UAM, Madrid, Spain, 2 Network Center for Biomedical Research in Neurodegenerative Diseases (CIBERNED), Madrid, Spain, 3 Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Madrid, Spain

Extracellular vesicles (EVs), like exosomes, play a critical role in physiological processes, including synaptic transmission and nerve regeneration. However, exosomes in particular can also contribute to the development of neurodegenerative conditions such as Alzheimer's disease (AD), Parkinson's disease, and prion diseases. All of these disorders are characterized by protein aggregation and deposition in specific regions of the brain. Several lines of evidence indicate that protein in exosomes is released from affected neurons and propagated along neuroanatomically connected regions of the brain, thus spreading the neurodegenerative disease. Also, different cell types contribute to the progression of tauopathy, such as microglia. Several groups have reported tau release via exosomes by cultured neurons or cells overexpressing human tau. Although the exact mechanisms underlying the propagation of protein aggregates are not fully understood, recent findings have implicated EVs in this process.  Both amyloid peptide and tau protein are present in specific exosomes. This review summarizes recent advances in our understanding of exosomes in the pathology of AD, with a special focus on tau protein.

#### Edited by:

Grant Thomas Corbett, Harvard Medical School, United States

#### Reviewed by:

Ioannis Sotiropoulos, University of Minho, Portugal Rostislav Skrabana, Institute of Neuroimmunology (SAS), Slovakia

#### \*Correspondence:

Félix Hernández<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Neurodegeneration, a section of the journal Frontiers in Neuroscience

Received: 05 April 2019 Accepted: 19 June 2019 Published: 02 July 2019

#### Citation:

Pérez M, Avila J and Hernández F (2019) Propagation of Tau via Extracellular Vesicles. Front. Neurosci. 13:698. doi: 10.3389/fnins.2019.00698 Keywords: tau propagation, extracellular vesicle, neurodegenerative disease, tau protein, Alzheimer's disease

#### INTRODUCTION

Brain microtubules were first isolated and characterized in the 1970s (Weisenberg, 1972). This study revealed the presence of a main protein, tubulin, and several others described as microtubuleassociated proteins (MAPs) in these structures. One of these MAPs, tau, was first characterized by Kirschner's group (Weingarten et al., 1975). Similar to other MAPs, the function of tau was found to be related to an increase in microtubule stabilization (Drubin and Kirschner, 1986), which prevents cell proliferation and facilitates neuronal differentiation. Recent discoveries about partners (End binding proteins 1 and 3, tRNA), different subcellular localizations (nucleus, nucleolus, plasma membrane, dendrites and dendritic spines) or association with cellular organelles (ribosomes, endoplasmic reticulum and the Golgi apparatus) for tau suggest additional roles. According to these studies, tau should be implicated on mechanisms of synaptic plasticity, structural architecture of heterochromatin, chromosome stability or regulating the cellular transcriptome (for more detail see review Sotiropoulos et al., 2017).

Although tau is mainly an intracellular protein, there are reports indicating that extracellular tau is present in brain interstitial fluid (Yamada et al., 2011), its amount decreasing in this medium during sleep (Lucey et al., 2019). Secreted tau may be implicated in some features of sleep (Cantero et al., 2010; Lucey et al., 2019). However, independently of this notion, secreted tau protein is present in vivo, and its secretion appears to be regulated.

### TAU SECRETION AND ITS REGULATION

The presence of extracellular tau suggests that it is secreted under physiological conditions. In this regard, it has been proposed that intracellular tau is released upon an increase in neuronal activity (Pooler et al., 2013; Yamada et al., 2014). In addition to neuron activity-dependent tau secretion, the extracellular form of the protein may arise by other mechanisms, such as neuron death (Gomez-Ramos et al., 2006), intracellular tau accumulation (Simon et al., 2012), a tauopathy (Clavaguera et al., 2009), or modulation by tau mutations (Karch et al., 2012). In the case of neuron death, intracellular proteins like tau are released into the extracellular space.