Aged Astrocytes Prime Brain for Neuroinflammation

Researchers believe that part of the reason why synapses disappear in neurodegenerative disease is that astrocytes and microglia reactivate synaptic pruning programs that had been dormant since development. However, few studies have looked at how astrocytes change in the healthy aging brain. Two recent mouse transcriptome studies start to fill this gap. In the February 7 Proceedings of the National Academy of Sciences, researchers led by Laura Clarke and the late Ben Barres at Stanford University School of Medicine reported that astrocytes in the aged mouse brain resembled the neuroinflammatory, lethal “A1” variety previously identified by the same lab (Jan 2017 news). Similarly, in the January 2 Cell Reports, researchers led by Nicola Allen at the Salk Institute for Biological Studies in La Jolla, California, documented an uptick in expression of inflammatory and synaptic pruning genes in aged astrocytes. Together, the papers suggest that elderly astrocytes enter a partially reactive state, perhaps helping explain why the aging brain is so susceptible to neurodegenerative disease. Both data sets are freely available to researchers online.

  • Two studies of astrocytes in aging mouse brain report similar results.
  • The cells take on a reactive phenotype, ramping up inflammatory and synaptic pruning genes.
  • This may prime the brain for inflammation and degeneration.

“We hope these resources will shed light on the mechanistic factors behind what happens in aging,” Clarke said. Allen, who previously worked in Barres’ lab, suggested that researchers studying neurodegenerative disease could use these data from healthy older brains as a baseline to dissect which expression changes result from disease and which are merely due to old age.

Commenters agree the data offer insights. “These are very important first steps in understanding how astrocytes evolve and how this evolution may facilitate the onset of aging-related disorders,” Benjamin Deneen at Baylor College of Medicine in Houston wrote to Alzforum (comment below). Anna Molofsky at the University of California, San Francisco, liked the focus on glia. “Glial cells in the brain, including astrocytes, are the first responders to most types of stress, and thus it is important to characterize how they may change their properties with age,” she wrote (comment below).

Previous studies had examined cultured astrocytes taken from aged rat brain, and found they overexpressed genes related to inflammation and oxidative stress compared with younger animals (Jiang and Cadenas, 2014; Bellaver et al., 2017). Others reported similar changes in astrocytes isolated from aged mouse brains via fluorescence-activated cell sorting (FACS) (Orre et al., 2014). However, these studies might not provide a full picture of what happens in brain, Allen noted. Glial cells are notorious for changing expression in culture, and FACS tears off fragile astrocytic processes, where much of the protein translation takes place.

First author Matthew Boisvert in Allen’s group instead used a ribosomal tag expressed only in astrocytes to selectively isolate these cells’ RNA from the brains of 4-month-old and 2-year-old mice. Boisvert and colleagues analyzed the visual and motor cortex, hypothalamus, and cerebellum, using three mice for each time point. In every region, expression went up for more genes than down. In general, genes expressed in reactive astrocytes such as GFAP, Serpina3n, and cytokines spiked, as did complement genes involved in synapse pruning, such as C3, C4b, and C1q. The resulting expression profile overlapped with that of reactive astrocytes, although the magnitude of the changes was smaller in the aged astrocytes.

Beyond this global aging signature, the authors also found regional differences in expression profiles. In cortical regions, about 50 transcripts changed with age, compared with  400–500 in the hypothalamus and cerebellum. Astrocytes in the cerebellum became the most reactive, those in cortical regions the least. The cerebellum loses neurons even during healthy aging, Allen noted. In addition, only cerebellar astrocytes accumulated the age-associated debris lipofuscin. “The findings raise the question: Does the cerebellum undergo accelerated aging?” she asked.

By contrast, the only change Allen and colleagues saw in the activity of astrocyte housekeeping genes was a drop in transcripts needed for cholesterol synthesis. In the brain, astrocytes supply most of the cholesterol, which is known to fall during aging, Allen noted. That may weaken synaptic function, she speculated, since cholesterol is an essential ingredient in neuronal membranes, including synaptic vesicles.

Rickie Patani at The Francis Crick Institute, London, was intrigued by the regional differences. “It is likely that a diverse spectrum of molecularly defined astrocyte reactive states will emerge,” he wrote to Alzforum. This makes sense, because astrocytes are already known to play numerous functional roles, he added.

Clarke and colleagues used methodology similar to Allen’s, isolating RNA from the hippocampus, striatum, and cortex of mouse brain in triplicate. They, too, used a ribosomal tag expressed only in astrocytes, albeit a different one. They examined several different time points, but focused their aging analysis on the comparison of 10-week-old mice and 2-year-old animals. Although they selected different brain regions than Allen and colleagues, their findings were similar. Aging astrocytes ramped up expression of inflammatory cytokines and complement genes, as well as genes involved in synaptic pruning like Mfge8 and Megf10. Few genes were expressed less, although the authors did see a drop in genes involved in energy production and antioxidant protection. This could contribute to metabolic and oxidative stress in aged brain, the authors suggested. They did not see a falloff in cholesterol synthesis genes, perhaps because they looked at different brain regions, Clarke noted.

Like Allen’s team, Clarke’s found regional differences, with cortical astrocytes displaying the fewest age-related changes. Meanwhile, most genes involved in basic astrocyte functions remained unchanged. “Our original hypothesis was that synaptogenic and phagocytic genes would change with age. We were surprised this was not the case,” Clarke told Alzforum.

Because the microglial cytokines IL1α, TNF, and C1qa prod astrocytes into the Al state, Clarke and colleagues analyzed aged mice lacking all three genes. They found a sharp reduction in expression of reactive astrocyte genes such as C3 and Cxcl10 in the knockouts compared to aged controls. This suggests that microglia stimulate many of the age-induced changes in astrocytes, they noted. Intriguingly, microglia have also been reported to enter a reactive state during aging (Grabert et al., 2016). Clarke plans to investigate whether ablating microglia in old mice and replacing them with microglia from young mice would ameliorate brain aging. However, she noted that aging astrocytes may trigger changes in microglia as well, creating a negative feedback loop. “We would like to figure out what initiates aging, and where we could best intervene,” Clarke said.

In addition, Clarke turned up evidence that age-related glial changes make the brain more prone to inflammation. Injecting lipopolysaccharides into aged mice to trigger an inflammatory response flipped more astrocytes into a reactive state than in young mice, Clarke noted.

How close are these mouse findings to what happens in human brain? Several studies have reported notable differences between rodent and human glia (Jan 2016 news; Jul 2017 news; Feb 2018 news). However, these studies also found an elevation of reactive genes, such as C3, in astrocytes from aged human brain, suggesting similar changes may occur in people, Clarke said. A team led by Patani and Jernej Ule at University College London previously reported that as human glia age, they become less regionally distinct (Jan 2017 news). “These findings [from Allen and Clarke] confirm and extend some of our recent work on human brain aging,” Patani wrote to Alzforum.

Allen suggested that while the mouse data may provide candidate aging genes, they will need to be validated in human tissue. Because brain samples of normally aging people are scarce, mouse brain provides a better system for initial gene discovery, she added.

Allen believes the findings from her and Clarke’s studies suggest that glial cells play a key role in brain aging. “If you want to understand diseases of aging, it will be important to study the whole brain and not just focus on neurons,” she noted.—Madolyn Bowman Rogers

Comments

    • User Profile Image Anna Victoria Molofsky
      UCSF Weill Institute for Neurosciences, University of California, San Francisco
    • Posted:

    Aging is associated with marked changes in brain function and is the biggest risk factor for the development of neurodegenerative diseases. Glial cells in the brain, including astrocytes, are the first responders to most types of stress, and thus it is important to characterize how they may change their properties with age. These two studies examine the changes in astrocyte gene expression between young and aged mice (equivalent perhaps to a 70-year-old human). Both agree on two key findings: 1) that different brain regions age in somewhat different ways, and 2) that aging, in general, is associated with similar gene expression changes as seen in reactive astrocytes—the type of astrocytes elicited after brain injury. Differences in the techniques used to purify transcripts in these two papers could account for some variation in results, but in general, these should serve as good resources to better understand how astrocyte functions change with age. A major question is the extent to which these gene expression changes correlate with changes in astrocyte functions, and whether those functions preserve or exacerbate age-related declines in brain health.

    View all comments by Anna Victoria Molofsky

  2. Over the past year, the nature of astrocyte diversity has emerged as a hot topic in the field of glial biology, with several papers describing both regional differences and distinct subpopulations in the normal brain (Chai et al., 2017; Morel et al., 2017; John Lin et al., 2017). Leveraging this knowledge, the two papers covered in this news story take the very important next steps and assess how region-specific astrocytes evolve over the course of aging.

    These are very important observations, as they show that astrocytes have a form of “plasticity”—meaning that they are not static populations of “glue,” but  rather exhibit a previously underappreciated responsiveness to their surrounding environment. These are indeed exciting times to be an astrocyte biologist, as these findings, coupled with the aforementioned studies that elucidated diverse astrocyte populations, point to the possibility that unique astrocyte "subpopulations" or "states”  demonstrate disease-specific associations.

    That the astrocytes found in the aged brain demonstrate immune “fingerprints” implicates them in aging-related brain disorders, like Alzheimer’s disease. Nevertheless, the prospective link between changed astrocytes in aging and bona fide degenerative disorders remains correlative, as much work remains to demonstrate actual causation. Admittedly, this is a major challenge because the cause-and-effect relationships between immune cells, astrocytes, neurons, and endothelial cells is not linear, but rather a web of interdependencies that occurs across equally complex and diverse degenerative disease states.

    In sum, these are very important first steps in understanding how astrocytes evolve and how this evolution may facilitate the onset of aging-related disorders.

    References:

    Chai H, Diaz-Castro B, Shigetomi E, Monte E, Octeau JC, Yu X, Cohn W, Rajendran PS, Vondriska TM, Whitelegge JP, Coppola G, Khakh BS. Neural Circuit-Specialized Astrocytes: Transcriptomic, Proteomic, Morphological, and Functional Evidence. Neuron. 2017 Aug 2;95(3):531-549.e9. Epub 2017 Jul 14 PubMed.

    Morel L, Chiang MS, Higashimori H, Shoneye T, Iyer LK, Yelick J, Tai A, Yang Y. Molecular and Functional Properties of Regional Astrocytes in the Adult Brain. J Neurosci. 2017 Sep 6;37(36):8706-8717. Epub 2017 Aug 7 PubMed.

    John Lin CC, Yu K, Hatcher A, Huang TW, Lee HK, Carlson J, Weston MC, Chen F, Zhang Y, Zhu W, Mohila CA, Ahmed N, Patel AJ, Arenkiel BR, Noebels JL, Creighton CJ, Deneen B. Identification of diverse astrocyte populations and their malignant analogs. Nat Neurosci. 2017 Mar;20(3):396-405. Epub 2017 Feb 6 PubMed.

    View all comments by Benjamin Deneen

  3. These papers provide useful information and an important resource. They also add further weight to the idea of a spectrum of astrocyte phenotypes, much like what has gradually appreciated with microglia ... which are not just active or resting, or even M1 or M2.

    Even during robust neurodegeneration, “reactive” astrocytes are not making their full secretory profile. We demonstrated “priming” of astrocytes, during neurodegeneration, to respond in an exaggerated fashion to acute cytokine stimulation (Hennessy et al., 2015). It will interesting to see to what extent this is true in aging and other degenerative models.

    References:

    Hennessy E, Griffin ÉW, Cunningham C. Astrocytes Are Primed by Chronic Neurodegeneration to Produce Exaggerated Chemokine and Cell Infiltration Responses to Acute Stimulation with the Cytokines IL-1β and TNF-α. J Neurosci. 2015 Jun 3;35(22):8411-22. PubMed.

    View all comments by Colm Cunningham

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References

News Citations

  1. Microglia Give Astrocytes License to Kill
  2. Purification of Adult Human Astrocytes Shows: They Are Unique
  3. Human and Mouse Microglia Look Alike, but Age Differently
  4. Microglial Transcriptome Hints at Shortcomings of AD Model
  5. Aging Causes “Identity Crisis” in Glia

Paper Citations

  1. Jiang T, Cadenas E. Astrocytic metabolic and inflammatory changes as a function of age. Aging Cell. 2014 Dec;13(6):1059-67. Epub 2014 Sep 19 PubMed.
  2. Bellaver B, Souza DG, Souza DO, Quincozes-Santos A. Hippocampal Astrocyte Cultures from Adult and Aged Rats Reproduce Changes in Glial Functionality Observed in the Aging Brain. Mol Neurobiol. 2017 May;54(4):2969-2985. Epub 2016 Mar 30 PubMed.
  3. Orre M, Kamphuis W, Osborn LM, Melief J, Kooijman L, Huitinga I, Klooster J, Bossers K, Hol EM. Acute isolation and transcriptome characterization of cortical astrocytes and microglia from young and aged mice. Neurobiol Aging. 2014 Jan;35(1):1-14. PubMed.
  4. Grabert K, Michoel T, Karavolos MH, Clohisey S, Baillie JK, Stevens MP, Freeman TC, Summers KM, McColl BW. Microglial brain region-dependent diversity and selective regional sensitivities to aging. Nat Neurosci. 2016 Mar;19(3):504-16. Epub 2016 Jan 18 PubMed.

Further Reading

Primary Papers

  1. Clarke LE, Liddelow SA, Chakraborty C, Münch AE, Heiman M, Barres BA. Normal aging induces A1-like astrocyte reactivity. Proc Natl Acad Sci U S A. 2018 Feb 20;115(8):E1896-E1905. Epub 2018 Feb 7 PubMed.
  2. Boisvert MM, Erikson GA, Shokhirev MN, Allen NJ. The Aging Astrocyte Transcriptome from Multiple Regions of the Mouse Brain. Cell Rep. 2018 Jan 2;22(1):269-285. PubMed.

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