23 Mar 2023

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 news; Jun 2021 news).

To directly assess how exosomes in the hippocampus relate to age, sex, and amyloidosis, first author Morgan Pait and colleagues used the in 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 news; Sep 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.

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.

“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.”—Jessica Shugart

Comments

1. • Tsuneya Ikezu
     Mayo Clinic Florida
   • Posted: 03 Apr 2023

   This is a technically challenging study to determine the actual composition of extracellular vesicles (EVs) in the interstitial fluid (ISF). The authors carefully conducted the experiment, and the proteomic results are exciting.

   The innovation is the use of in vivo microdialysis to collect and monitor EVs from the hippocampal ISF of live mice. They applied this method to isolate ISF EVs from APP/PS1 mice of different ages and sexes and characterized the EV proteomics. The proteins in their ISF EVs showed some overlap with our previous EV dataset from CAST_APPPS1 mouse brain (Muraoka et al., 2021). By examining the cell-type-specific signatures of ISF EVs, they found that microglial EV proteins were most significantly correlated with Aβ pathology. They also observed a sexual dimorphism in the ISF EV proteome, suggesting that female APP/PS1 mice may respond differently to Aβ deposition from APP/PS1 males. The data suggest that microglial-derived EVs could serve as an attractive early biomarker of Aβ-related changes in the brain.

   Our more recent proteomic profiling of brain-derived EVs isolated from AD, MCI controls show enrichment of activated astrocytes in AD cases compared to MCI or control cases (You et al., 2022).

   One limitation of this study is the potential low EV yield from the microdialysis, and the possibility that the cell-type enrichment of EVs may be artificially affected by the location of insertion of the probe into the brain. Another consideration is the potential complication by the surgery to cause inflammatory responses in the mouse brain, which could alter the EV cargo composition, especially those of microglial EVs. The study is nonetheless very interesting and will provide resource for our understanding of CNS EV biology in healthy and disease conditions.

   Yang You, Ph.D., of the Mayo Clinic is a co-author of this comment.

   References:

   Muraoka S, Jedrychowski MP, Iwahara N, Abdullah M, Onos KD, Keezer KJ, Hu J, Ikezu S, Howell GR, Gygi SP, Ikezu T. Enrichment of Neurodegenerative Microglia Signature in Brain-Derived Extracellular Vesicles Isolated from Alzheimer's Disease Mouse Models. J Proteome Res. 2021 Mar 5;20(3):1733-1743. Epub 2021 Feb 3 PubMed.

   You Y, Muraoka S, Jedrychowski MP, Hu J, McQuade AK, Young-Pearse T, Aslebagh R, Shaffer SA, Gygi SP, Blurton-Jones M, Poon WW, Ikezu T. Human neural cell type-specific extracellular vesicle proteome defines disease-related molecules associated with activated astrocytes in Alzheimer's disease brain. J Extracell Vesicles. 2022 Jan;11(1):e12183. PubMed.

   View all comments by Tsuneya Ikezu

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References

News Citations

1. Exosomes Stand Out as Potential Blood Biomarkers 1 Aug 2014
2. Exosomes Divulge Lysosomal Unrest, Signal AD? 15 Jun 2015
3. Neuronal Exosomes Embroiled in Controversy 10 Jun 2021
4. Soluble Aβ: Getting a Grip on Its Fate 9 Oct 2003
5. Brain Microdialysis Reveals Tau, Synuclein Outside of Cells 23 Sep 2011

Further Reading

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