Normal aging induces A1-like astrocyte reactivity PNAS PLUS Laura E. Clarkea,1, Shane A. Liddelowa,b,c, Chandrani Chakrabortya, Alexandra E. Müncha, Myriam Heimand,e,f, and Ben A. Barresa,2 aDepartment of Neurobiology, Stanford University, School of Medicine, Stanford, CA 94305; bNeuroscience Institute, NYU Langone Medical Center, New York, NY 10016; cDepartment of Neuroscience and Physiology, NYU Langone Medical Center, New York, NY 10016; dBroad Institute of MIT and Harvard, Cambridge, MA 02142; ePicower Institute for Learning and Memory, Cambridge, MA 02139; and fDepartment of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139 Edited by Carla J. Shatz, Stanford University, Stanford, CA, and approved January 19, 2018 (received for review December 12, 2017) The decline of cognitive function occurs with aging, but the activated morphology in aged brains (17). The extent to which mechanisms responsible are unknown. Astrocytes instruct the astrocytes also become activated with age is unknown, although formation, maturation, and elimination of synapses, and impair- it is reported that the level of glial fibrillary acidic protein ment of these functions has been implicated in many diseases. (GFAP) and vimentin, two astrocyte-specific genes linked to These findings raise the question of whether astrocyte dysfunc- activation, increase with age in both rodents and humans (18, tion could contribute to cognitive decline in aging. We used the 19). Regardless, the transcriptional and functional changes oc- Bac-Trap method to perform RNA sequencing of astrocytes from curring in astrocytes in normal aging are not well understood. different brain regions across the lifespan of the mouse. We found The up-regulation of potentially proinflammatory genes by that astrocytes have region-specific transcriptional identities that microglia in normal aging raises the question of whether these change with age in a region-dependent manner. We validated our aging-induced changes in microglia could promote the activation of findings using fluorescence in situ hybridization and quantitative astrocytes. Recently, we reported that proinflammatory microglia PCR. Detailed analysis of the differentially expressed genes in secrete IL-1α, TNF, and C1q, and that these cytokines acting to- aging revealed that aged astrocytes take on a reactive phenotype gether are necessary and sufficient to activate astrocytes (9). These of neuroinflammatory A1-like reactive astrocytes. Hippocampal cytokines induce the formation of a subtype of astrocytes (termed and striatal astrocytes up-regulated a greater number of reactive A1 astrocytes) which are strongly neurotoxic and rapidly kill neu- astrocyte genes compared with cortical astrocytes. Moreover, aged brains formed many more A1 reactive astrocytes in response rons (9). As well as releasing a potent neurotoxin, A1 astrocytes NEUROSCIENCE to the neuroinflammation inducer lipopolysaccharide. We found were less able to promote the formation of new synapses, and that the aging-induced up-regulation of reactive astrocyte genes caused a decrease in the excitatory function of CNS neurons. In was significantly reduced in mice lacking the microglial-secreted addition to changes in the reactive state of astrocytes, it is possible cytokines (IL-1α, TNF, and C1q) known to induce A1 reactive astro- that other transcriptional and functional changes are occurring in cyte formation, indicating that microglia promote astrocyte acti- astrocytes that contribute to cognitive decline in normal aging. vation in aging. Since A1 reactive astrocytes lose the ability to To investigate the transcriptional changes occurring in astro- carry out their normal functions, produce complement compo- cytes with aging, we performed RNA sequencing (RNAseq) at five nents, and release a toxic factor which kills neurons and oligoden- ages across the lifespan of the mouse to determine the aging-induced drocytes, the aging-induced up-regulation of reactive genes by astrocytes could contribute to the cognitive decline in vulnerable Significance brain regions in normal aging and contribute to the greater vul- nerability of the aged brain to injury. In aging, the brain becomes vulnerable to injury and cognitive function declines, but the mechanisms responsible are un- astrocytes | aging | microglia | RNA sequencing | cognitive decline known. Astrocytes, the most abundant class of glial cells, are vital for the proper function of the central nervous system, and strocytes are the most abundant glial cells and are vital for impairment of astrocyte function has been implicated in dis- Athe proper function of the central nervous system (CNS). ease. Here we perform RNA sequencing of astrocytes from They perform many roles, including instructing the formation (1– different brain regions across the lifespan of the mouse to 3) and elimination (4) of neuronal synapses during development. identify age-related transcriptional changes that could con- In addition, they provide trophic support to neurons (5), mediate tribute to cognitive decline. We find that aged astrocytes take uptake and recycling of neurotransmitters (6), and are involved on a reactive phenotype characteristic of neuroinflammatory in maintenance of the blood–brain barrier (7). Given the mul- reactive astrocytes, and that microglia play a role in inducing titude of vital roles for astrocytes, it is not surprising that im- astrocyte activation. The aging astrocyte RNA sequencing pairment of these functions has been implicated in the profiles provide an important new resource for future studies pathogenesis of many diseases. In particular, astrocyte dysfunc- exploring the role of astrocytes in cognitive decline. tion has been linked to age-related neurodegenerative diseases, including Alzheimer’sdisease(8–10), dementia (11), and amyo- Author contributions: L.E.C. and B.A.B. designed research; L.E.C., S.A.L., C.C., A.E.M., and M.H. performed research; M.H. contributed new reagents/analytic tools; L.E.C. and S.A.L. trophic lateral sclerosis (ALS) (12, 13). Several hallmarks of analyzed data; and L.E.C. and B.A.B. wrote the paper. these neurodegenerative diseases, such as cognitive decline Conflict of interest statement: B.A.B. was a cofounder of Annexon Biosciences Inc., a and a loss of working memory, also occur during the process company working to make new drugs for treatment of neurological diseases. of normal aging. This article is a PNAS Direct Submission. Transcriptomics and epigenetic studies have reported that This open access article is distributed under Creative Commons Attribution-NonCommercial- glial-specific gene expression patterns change significantly with NoDerivatives License 4.0 (CC BY-NC-ND). normal aging, while neuron-specific gene expression patterns Data deposition: The data reported in this paper have been deposited in the BioProject change relatively little in comparison (14). For instance, micro- database, https://www.ncbi.nlm.nih.gov/bioproject (accession no. PRJNA417856). glia, the resident immune cells of brain, up-regulate immune 1To whom correspondence should be addressed. Email: [email protected]. response signaling receptors (15), and neuroprotective signaling 2Deceased December 27, 2017. pathways (16) in aged mice. Consistent with the observed tran- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. scriptional changes, there is evidence that microglia adopt an 1073/pnas.1800165115/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1800165115 PNAS Latest Articles | 1of10 Downloaded by guest on September 30, 2021 transcriptional changes occurring in astrocytes. Furthermore, we phenotype in different brain regions (hippocampus, striatum, profiled astrocytes from three functionally distinct brain and cortex) we generated an RNAseq profiling resource of regions—the hippocampus, striatum and cortex—at these five adolescent [postnatal day 7 (P7)], young adult (P32), mature ages, to investigate brain-region−specific changes in the astrocyte (10 wk), middle age (9.5 mo), and aged (2 y) astrocytes. A major transcriptome in normal aging. Our RNAseq data revealed that limitation to sequencing older CNS tissues is the successful pu- astrocytes have distinct region-dependent transcriptional identi- rification of intact, viable astrocytes with minimal isolation arti- ties and age in a regionally dependent manner. In particular, we fact. To overcome this difficulty, we used the Aldh1l1-eGFP- found that astrocytes in regions more vulnerable to cognitive L10a transgenic mouse line (Fig. 1A), which expresses a fusion of decline—the hippocampus and striatum—undergo more dra- eGFP and a ribosomal protein (L10a) under the control of the matic transcriptional changes compared with cortical astrocytes. astrocyte-specific Aldh1l1 gene. Translating ribosome affinity Differential gene expression analysis revealed that the most purification (TRAP) (21, 22) of this fusion protein yields the prominent class of genes up-regulated by aged astrocytes were ribosome and its associated mRNAs, allowing the isolation of reactive astrocyte genes. Interestingly, many of these reactive transcripts from the eGFP-expressing astrocytes. To first assess astrocyte genes are characteristic A1 reactive astrocyte genes that the specificity of ALDH1L1-eGFP-L10a protein expression in are induced in response to neuroinflammation (9, 20). We found adult mice (10 wk of age), we performed immunohistochemistry that the aging-induced up-regulation of