Elsevier

Neurobiology of Aging

Volume 35, Issue 8, August 2014, Pages 1792-1800
Neurobiology of Aging

Regular article
Exosome reduction in vivo is associated with lower amyloid plaque load in the 5XFAD mouse model of Alzheimer's disease

https://doi.org/10.1016/j.neurobiolaging.2014.02.012Get rights and content

Abstract

We present evidence here that exosomes stimulate aggregation of amyloid beta (Aβ)1−42 in vitro and in vivo and interfere with uptake of Aβ by primary cultured astrocytes and microglia in vitro. Exosome secretion is prevented by the inhibition of neutral sphingomyelinase 2 (nSMase2), a key regulatory enzyme generating ceramide from sphingomyelin, with GW4869. Using the 5XFAD mouse, we show that intraperitoneal injection of GW4869 reduces the levels of brain and serum exosomes, brain ceramide, and Aβ1−42 plaque load. Reduction of total Aβ1–42 as well as number of plaques in brain sections was significantly greater (40% reduction) in male than female mice. Our results suggest that GW4869 reduces amyloid plaque formation in vivo by preventing exosome secretion and identifies nSMase2 as a potential drug target in AD by interfering with exosome secretion.

Introduction

Alzheimer's disease (AD) is characterized by cognitive decline, alteration of synaptic transmission, and neuronal death brought on by accumulation of neurofibrillary tangles in neurons and amyloid beta (Aβ) in the brain parenchyma. There is no viable therapy to slow or reverse AD progression. The leading hypothesis for the cause of AD is an impairment of clearance of Aβ resulting from the β-cleavage of the amyloid precursor protein (APP) (Bertram and Tanzi, 2008). Strong evidence comes from early-onset familial AD, in which patients carry mutations in genes encoding for APP or the presenilin-1 (PS1) component of the γ-secretase complex that cleaves APP to generate Aβ (Bird, 2008, Selkoe, 2011). Recent studies showing that reducing Aβ load in mouse models improves cognitive function strongly suggest that reducing plaque burden is a potential treatment strategy for AD (Cramer et al., 2012, Geekiyanage et al., 2013, Liu et al., 2013).

Exosomes are ceramide-enriched vesicles, 40–100 nm in diameter, generated by inward budding of the endosomal membrane, and secreted when these multivesicular endosomes fuse with the plasma membrane (Thery, 2011, Trajkovic et al., 2008). Exosomes carry signaling factors and microRNAs that mediate intercellular communication, and there is evidence that exosomes play a role in the progression of AD (Bellingham et al., 2012). Aβ processing has been shown to be associated with endosomal compartments with a fraction of Aβ and C-terminal fragments being secreted in association with exosomes (Haass et al., 1995, Perez-Gonzalez et al., 2012, Rajendran et al., 2006, Sharples et al., 2008, Vassar et al., 1999). It is however unclear what role exosomes play in the aggregation and/or clearance of Aβ. Exosomes have been implicated in the extracellular enzymatic degradation of Aβ (Bulloj et al., 2010) and in contrast, are reported to promote Aβ fibrillization and clearance by microglia (Yuyama et al., 2012). Our laboratory reported that cultured primary astrocytes secrete exosomes in response to Aβ exposure, a process dependent upon ceramide generation by neutral sphingomyelinase 2 (nSMase2) (Wang et al., 2012), which suggests that exosome secretion is upregulated during AD. Other work has shown that glia preferentially take up oligomeric Aβ compared with fibrils (Nielsen et al., 2010), suggesting that Aβ aggregation interferes with efficient clearance. In this study, we show that exosomes promote Aβ aggregation and by extension, plaque formation in vivo. Inhibition of nSMase2 with GW4869 (Luberto et al., 2002) in the 5XFAD mouse blocks exosome secretion to reduce plaque formation and decrease the overall brain amyloid load.

Section snippets

Animals and GW4869 administration

All experiments involving animals followed approved protocols by Georgia Regents University's Institutional Animal Care and Use Committee. Mice expressing 5 human mutations in APP and PS1 (B6SJL-Tg[APP*K670N*M671L*I716V*V717I, PSEN1*M146*L286V]6799Vas/J) under neuron-specific elements of the Thy1 promoter were purchased from The Jackson Laboratory and crossed to wild-type C57Bl/6 mice to generate offspring hemizygous for the APP and PS1 transgenes. These mice predominately generate Aβ1–42 that

Astrocyte-derived exosomes promote Aβ aggregation and interfere with uptake by glia

Because exosomes derived from N2a neuroblastoma cells were able to promote Aβ aggregation in vitro (Yuyama et al., 2012), we tested whether astrocyte-derived exosomes held this property. Monomeric Aβ1–42 (20 μM) was incubated at 37 °C for 18 hours with or without exosomes collected from astrocyte-conditioned medium. Exosomes stimulated the aggregation of Aβ, visible as a pellet following centrifugation, approximately 20-fold over Aβ incubated alone. Inclusion of rabbit anti-ceramide IgG (

Discussion

The present study reveals nSMase2 as a novel potential therapeutic target in the treatment of AD. One of the hallmarks of AD progression is chronic neuroinflammation (Broussard et al., 2012). Amyloid peptides activate astrocytes and microglia, which secrete pro-inflammatory cytokines such as IL-1β and TNF-α (Meda et al., 1995, Sheng et al., 1996) that mediate activation of SMases to generate ceramide, which can lead to apoptosis (Ariga et al., 1998, Rybakina et al., 2001). Moreover, nSMase

Disclosure statement

The authors declare that they have no actual or potential conflicts of interest. All experiments involving animals followed approved protocols by Georgia Regents University's Institutional Animal Care and Use Committee.

Acknowledgements

This work was funded by the National Institutes of Health R01-AG034389 to E.B. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors thank C. Wakade and J. Pihkala for additional technical assistance. They are grateful to Katarzyrna Pituch (supervisor Dr Irene Givogri), University of Illinois at Chicago, for assistance with the exosome isolation from brain. They thank the imaging core facility (under supervision

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