Border Surprise: Glia Limitans Astrocytes Sit on Brain Surface

In their growing interest in the brain's border tissues, Alzheimerologists realize that they know little about some of its cells. One new study now sheds light on the astrocytes that form the glia limitans, which separates the brain's parenchyma from its meninges. Scientists led by Philip Hasel and Shane Liddelow at New York University used a combination of single-cell RNA-Seq and spatial transcriptomics to home in on these cells. In a preprint on bioRxiv, they report that glia limitans astrocytes are highly specialized, with a unique transcriptional profile that includes many reactivity genes and is characterized by the extracellular matrix protein myocilin.  

  • Glia limitans astrocytes are marked by expression of ECM protein myocilin. 
  • They express many reactivity proteins, perhaps to guard the brain from outside insults. 
  • In a surprise, it's their cell bodies, not their end feet, that sit on the brain’s surface.

 

The biggest surprise was where these cells are. Contrary to conventional wisdom, which held that glia limitans astrocytes reside in the parenchyma and extend end feet upward to form the barrier, the authors located their cell bodies on the brain’s surface, with their processes extending down into the parenchyma. If confirmed, these findings lay the groundwork for further study of how glia limitans astrocytes protect the brain, the authors suggested. 

Michael Sofroniew at the University of California, Los Angeles, agreed. “The glia limitans has largely been neglected in experimental studies,” he told Alzforum. “New data from this and other studies are beginning to generate a molecular profile. This represents a major advance.”

Sitting on Top. In mouse brain (left), glia limitans astrocytes (white) sit on the surface and extend processes downward. Cortical blood vessels (right) nestle in grooves formed by these astrocytes, with processes (thin red lines) crossing the vessels (false color scale represents imaging depth). [Courtesy of Hasel et al., 2023.] 

Previous scRNA-Seq studies have enabled researchers to identify multiple distinct states for microglia and to subtype neurons to a greater degree than previous methods did. However, astrocytes tend to be underrepresented in these studies, with many datasets including only about 1,000 astrocytes total. This means rare or spatially restricted astrocyte subtypes stay under the radar, and other techniques must be used to detect them. Recently, Hasel and Liddelow combined scRNA-Seq and spatial transcriptomics to identify a rare subtype of inflammatory astrocyte (Aug 2022 conference news). They wondered if this method also could provide more information about the astrocytes that encase the brain. 

Hasel plumbed existing data, first reanalyzing the lab’s previous spatial transcriptomics dataset from adult mouse brain. He found a distinct gene expression profile that distinguished cells on the surface of the brain, and also separated the cortex from subcortical regions. To find out what cells these were, Hasel examined a scRNA-Seq dataset generated by researchers led by Lee Rubin at Harvard (Ximerakis et al., 2019). The brain-surface profile was specific to astrocytes, and was characterized by the expression of myocilin. This ECM protein is best known for forming part of the trabecular meshwork, a series of tiny canals in the eye that drain excess fluid. Mutations in myocilin cause glaucoma.  

Myocilin+ cells were rare, making up only 2 percent of all astrocytes. These cells also expressed many genes involved in immune reactivity, such as IFITM3, A2M, and C4B, which other astrocytes did not. Potentially, these reactivity genes could allow glia limitans astrocytes to respond to peripheral inflammatory signals, the authors suggested. They believe the cells may act as sentinels that guard the brain. 

Hasel and colleagues used in situ hybridization to confirm that myocilin+ cells were indeed astrocytes and were confined to the brain’s surface. To get a closer look, the authors cleared away lipids with a commercial protocol called Adipo-Clear, leaving the tissue transparent (Chi et al., 2018). In reporter mice with fluorescent astrocytes, they now saw myocilin+ cell bodies lining the brain surface. The cells were flat, with few processes, in contrast to the bushy shapes of parenchymal astrocytes. Blood vessels running along the cortical surface nestled into grooves in this astrocyte layer, with astrocyte processes crossing these vessels like netting (see image above). Potentially, glia limitans astrocytes contribute to the brain’s structural integrity, the authors suggested. 

Some previous studies have also found astrocyte cell bodies on the brain’s surface (Feig and Haberly, 2011; Falcone et al., 2021), and one reported these cells were myocilin+ (Wu et al., 2017).

Human Brain, Too. In postmortem brain samples, myocilin+ astrocytes (purple) cover the brain surface and extend processes into the parenchyma. [Courtesy of Hasel et al., 2023.]

Do these astrocytes also cover the human brain? The authors analyzed single-nuclei RNA-Seq datasets from their and other labs, finding a myocilin+ gene expression profile similar to that seen in mice (Sadick et al., 2022; Siletti et al., 2023). In the much larger human brain, only 0.13 percent of astrocytes were myocilin+, suggesting this cell state would be missed in most studies. In postmortem human brain samples, immunostaining for myocilin again revealed the cells on the surface, extending their processes downward (see image above). 

Hasel is generating transgenic mice that will allow him to express genes specifically in myocilin+ astrocytes. In future work, he plans to use these mice to study the cells’ function, including whether they form a barrier that keeps out peripheral cells and molecules. 

Meanwhile, Sofroniew has a preprint on bioRxiv showing similarities between glia limitans astrocytes and the astrocytes that proliferate to form scars around brain lesions (O’Shea et al., 2023). “We think these ‘wound repair’ astrocytes are re-establishing borders that separate CNS neural tissue from non-neural tissue, and this is similar to the separation between neural tissue and meninges achieved by glia limitans astrocytes,” Sofroniew wrote to Alzforum. Wound-repair astrocytes do not express myocilin, however. This gene seems to be unique to the glia limitans.—Madolyn Bowman Rogers

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References

News Citations

  1. High-Res Spatial Transcriptomics Offers New Views of Mouse Brain

Paper Citations

  1. Ximerakis M, Lipnick SL, Innes BT, Simmons SK, Adiconis X, Dionne D, Mayweather BA, Nguyen L, Niziolek Z, Ozek C, Butty VL, Isserlin R, Buchanan SM, Levine SS, Regev A, Bader GD, Levin JZ, Rubin LL. Single-cell transcriptomic profiling of the aging mouse brain. Nat Neurosci. 2019 Oct;22(10):1696-1708. Epub 2019 Sep 24 PubMed.
  2. Chi J, Wu Z, Choi CH, Nguyen L, Tegegne S, Ackerman SE, Crane A, Marchildon F, Tessier-Lavigne M, Cohen P. Three-Dimensional Adipose Tissue Imaging Reveals Regional Variation in Beige Fat Biogenesis and PRDM16-Dependent Sympathetic Neurite Density. Cell Metab. 2018 Jan 9;27(1):226-236.e3. PubMed.
  3. Feig SL, Haberly LB. Surface-associated astrocytes, not endfeet, form the glia limitans in posterior piriform cortex and have a spatially distributed, not a domain, organization. J Comp Neurol. 2011 Jul 1;519(10):1952-69. PubMed.
  4. Falcone C, Penna E, Hong T, Tarantal AF, Hof PR, Hopkins WD, Sherwood CC, Noctor SC, Martínez-Cerdeño V. Cortical Interlaminar Astrocytes Are Generated Prenatally, Mature Postnatally, and Express Unique Markers in Human and Nonhuman Primates. Cereb Cortex. 2021 Jan 1;31(1):379-395. PubMed.
  5. Wu YE, Pan L, Zuo Y, Li X, Hong W. Detecting Activated Cell Populations Using Single-Cell RNA-Seq. Neuron. 2017 Oct 11;96(2):313-329.e6. PubMed.
  6. Sadick JS, O'Dea MR, Hasel P, Dykstra T, Faustin A, Liddelow SA. Astrocytes and oligodendrocytes undergo subtype-specific transcriptional changes in Alzheimer's disease. Neuron. 2022 Jun 1;110(11):1788-1805.e10. Epub 2022 Apr 4 PubMed.
  7. Siletti K, Hodge R, Mossi Albiach A, Lee KW, Ding SL, Hu L, Lönnerberg P, Bakken T, Casper T, Clark M, Dee N, Gloe J, Hirschstein D, Shapovalova NV, Keene CD, Nyhus J, Tung H, Yanny AM, Arenas E, Lein ES, Linnarsson S. Transcriptomic diversity of cell types across the adult human brain. Science. 2023 Oct 13;382(6667):eadd7046. PubMed.
  8. O'Shea TM, Ao Y, Wang S, Ren Y, Cheng A, Kawaguchi R, Swarup V, Sofroniew MV. Border-forming wound repair astrocytes. 2023 Aug 27 10.1101/2023.08.25.554857 (version 1) bioRxiv.

Further Reading

Primary Papers

  1. Hasel P, Cooper ML, Marchildon AE, Rufen-Blanchette U, Kim RD, Ma TC, Kang UJ, Chao MV, Liddelow SA. Defining the molecular identity and morphology of glia limitans superficialis astrocytes in mouse and human. 2023 Apr 06 10.1101/2023.04.06.535893 (version 1) bioRxiv.
  2. O'Shea TM, Ao Y, Wang S, Ren Y, Cheng A, Kawaguchi R, Swarup V, Sofroniew MV. Border-forming wound repair astrocytes. 2023 Aug 27 10.1101/2023.08.25.554857 (version 1) bioRxiv.

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