Exosomes from living mice hint that sexes respond differently to amyloid

As part of the myriad modes of communication within the brain, cells can secrete tiny extracellular vesicles, each carrying a cadre of molecules that reflect the cell’s state. Scientists have now devised a method to intercept these vesicular packages within a living mouse. As described in a bioRxiv preprint posted March 12, researchers led by Gagan Deep and Shannon Macauley of Wake Forest School of Medicine in Winston-Salem, North Carolina, used microdialysis to sample extracellular vesicles (EVs) from the interstitial fluid of the hippocampus, even as animals roamed freely in their cages. In a model of amyloidosis, the protein contents of the vesicular packets—particularly those churned out by microglia— varied markedly by age, sex, and the extent of amyloid deposition. Though female mice accumulated twice as much amyloid as their male counterparts, their microglia appeared to release fewer EVs containing fewer proteins. The findings suggest that exosomal contents are tied to microglial function, and might be useful disease biomarkers.

EVs carry a mix of RNAs, proteins, and metabolites that reflect the type and state of their parent cell. A tiny type of EV, exosomes originate from endosomes that fuse with the plasma membrane. Brain-derived exosomes can be found within the cerebrospinal fluid and plasma, and some researchers view them as a potential trove of biomarkers (Aug 2014 conference news). However, it has been difficult to determine how faithfully they reflect ongoing events within different parts of the brain (Jun 2015 newsJun 2021 news).

To directly assess how exosomes in the hippocampus relate to age, sex, and amyloidosis, first author Morgan Pait and colleagues used the vivo microdialysis method (image above). The technique has previously been used to study the ebb and flow of proteins, including Aβ, tau, α-synuclein, and ApoE, within brain fluid (Oct 2003 newsSep 2011 news). The scientists inserted a guide cannula directly into the left hippocampus, through which they inserted a probe equipped with a 1,000kDa cut-off filter. Pumping in artificial cerebrospinal fluid, ISF was slowly collected every 90 minutes over a period of 72 hours as the mice roamed freely, ultimately yielding enough fluid to pellet small EVs, including exosomes, via ultracentrifugation. The researchers then deployed flow cytometry and mass spectrometry to measure the number, size, and proteomes of the EVs. They analyzed EVs from wild-type mice, and from APP/PS1 mice, at 3 and 9 months of age. In the latter, that’s before and after Aβ plaques grew in the brain. Notably, by 9 months, female APP/PS1 mice had around double the load of amyloid plaques as males.

Exosomal Interception. Using a microdialysis probe inserted into the hippocampus, scientists gently pumped in artificial cerebrospinal fluid while extracting interstitial fluid, from which they isolated extracellular vesicles. Mice roamed freely during the sampling process. [Courtesy of Plait et al., bioRxiv, 2023.]

As male wild-type or APP/PS1 mice aged, the concentration of EVs within their hippocampal ISF increased by about two orders of magnitude. For females, the concentration of EVs in the ISF held steady, regardless of age or genotype.

“We were very surprised to see such a strong sex-specific difference in EV concentration with age,” Macauley told Alzforum.

For mice of both sexes and genotypes, the concentration of proteins within their EVs increased with age. Proteomic analysis of ISF EVs identified 436 proteins involved in myriad biological processes, including proteostasis, inflammation, immune function, metabolism, and cellular structural integrity. Of these, 168 were shared across mice of both genotypes and both ages, pointing to a core group of hippocampal EV proteins. Smaller numbers of proteins were unique to each genotype and age. Interestingly, although the concentration of total proteins increased with age regardless of genotype, the diversity of proteins dropped with older age and APP/PS1 genotype. In other words, 3-month-old wild-type mice had a larger variety of proteins in their EVs than their 9-month-old counterparts, and APP/PS1 vesicles had less variety regardless of age. The number of biological processes in which the EV proteins were involved was also lower in APP/PS1 mice relative to wild-type animals.

Hippocampal EV Repertoire. Of the 436 proteins identified across mice of both ages and genotypes, 168 were shared by EVs from all groups. Far fewer proteins were unique to any given age or genotype. [Courtesy of Pait et al., bioRxiv, 2023.]

As wild-type mice aged, their hippocampal exosomes included more proteins involved in proteostasis and immune function, while these types of proteins increased less so with age among APP/PS1 exosomes. Notably, immune proteins were elevated in exosomes from 3-month-old APP/PS1 mice relative to young wild-type mice, suggesting an immune response mounting ahead of plaque deposition, but then not increasing later on.

How did exosomes secreted by different cell types change with age, sex, and genotype? To find out, the scientists used cell type markers to trace the cellular origin of exosomes. The standout findings came from microglia. Numbers of EVs bearing microglial markers increased in step with amyloid deposition in male but not female mice, suggesting that in males microglia responded differently to Aβ deposition than in females. Notably, microglial exosomes from 9-month-old APP/PS1 females, but not males, contained apolipoprotein E and clusterin, two proteins implicated in AD pathogenesis. These exosomal distinctions between the sexes occurred in parallel with broader differences in how microglia responded to plaques. While microglia in both male and female mice increased their expression of the activation marker CD68 as plaques grew, microglia in male mice gathered more intensely around nascent plaques at 3 months of age, suggesting male microglia mounted an early, more specific response to amyloidosis.

Zeroing in on Plaques. At 3 months of age, microglia (green) in female mice (top) homed less efficiently to amyloid plaques (blue) than microglia in male mice (bottom), which co-localized more tightly with plaques. [Courtesy of Plait et al., bioRxiv, 2023.]

“The study using in vivo microdialysis is a very powerful strategy to capture directly what is happening in the brain, as shown here for sex-dependent changes in the microglial proteome of extracellular vesicles in a model of amyloidosis,” wrote Jürgen Götz of the University of Queensland in Australia.

Still, Götz believes that most labs working in this area of research will continue to rely on more conventional methods of isolating extracellular vesicles, such as differential centrifugation of brain samples.

Macauley hopes the findings will be of use for biomarker development.

“If we can define distinct signatures of extracellular vesicles at the site of AD pathology, this will point to exosomal signatures to look for in the CSF or blood of people with Alzheimer’s,” she said.

Importantly, the findings also suggest that microglial, as opposed to neuronal, exosomes might be the most responsive biomarkers to AD pathogenesis.

Celeste Karch of Washington University in St. Louis agreed. “Most studies use neuron- or astrocyte-derived EVs/exosomes in human studies as biomarkers of AD progression, but this study shows that neuron-derived EVs may not serve as the best biomarker. Microglia-derived EVs track more closely with amyloidosis.”


Pait MC, Kaye SD, Su Y, Kumar A, Singh S, Gironda SC, Vincent S, Anwar M, Carroll CM, J. Snipes A, Lee J, Furdui CM, Deep G, Macauley SL. (2023) Novel method for collecting hippocampal interstitial fluid extracellular vesicles (EVISF) reveals sex-dependent changes in microglial EV proteome in response to Aβ pathology. bioRXiv [online preprint]. [article]

Leave a Reply

Your email address will not be published. Required fields are marked *