When Autophagy Stops, Microglia Sour into Senescence

When deprived of their ability to dispose of detritus via autophagy, microglia become annoyed, transitioning into a senescent, dysfunctional state. That was the upshot of a study published May 25 in Nature Cell Biology. Zhenyu Yue at the Icahn School of Medicine at Mount Sinai, New York, and colleagues used conditional knockout mice to disable the “self-eating” pathway in microglia. As if in a fit of pique, some of the cells shut down their cell cycle and revved up secretion of cytokines—behavior typical of senescent cells. In amyloid-laden mice, these autophagy-deficient microglia refused to transition into a bona fide disease-associated (DAM) state, failing to properly contain plaques or to protect nearby synapses from shriveling. Treating these mice with senolytic drugs assuaged some of these refuseniks. The findings further raise the profile of microglial autophagy as an essential part of the brain’s response to proteopathic insults.

  • Microglia deficient in autophagy failed at amyloid plaque containment, neuroprotection.
  • They took on a senescent rather than a disease-associated state.
  • A senolytic drug cocktail rescued DAM microglia, and suppressed plaque buildup.

Neurons suffer dramatic impairments in autophagy both in the AD brain and in mouse models of amyloidosis (Nixon et al., 2005; Jun 2010 news; Jun 2022 news). While neurons need autophagy to clean up their own waste, microglia need this pathway for an additional purpose, that is, to help them mop up protein aggregates and detritus spewed by sickly neurons. Recent studies have cast microglial autophagy—a bioenergetically demanding process—as quelling Aβ plaques, tau pathology, and neuroinflammation (Aug 2017 news; Aug 2020 news; Xu et al., 2021).

When faced with amyloid accumulation, microglia, particularly those in mouse models of amyloidosis, transform into DAM, a transcriptional state associated with phagocytosis and other protective functions (Jun 2017 news; Sep 2022 conference news). How does autophagy relate to this microglial metamorphosis? First author Insup Choi and colleagues investigated.

First, they noticed that in plaque-laden 5xFAD mice, microglia that had transitioned into the DAM state—as indicated by elevated expression of Clec7a—had also dialed up their autophagy. DAM contained an abundance of double-membraned autophagosomes and expressed a bevy of other autophagic markers.

How would these cells behave without autophagy? To find out, the researchers conditionally deleted, from wild-type mice, the Atg7 gene, which encodes a protein critical for autophagosome biogenesis, only from microglia. They knocked out Atg7 in 2-month-old mice, then examined their brains six months later. Using single-cell transcriptomics, the scientists detected eight gene-expression clusters of microglia in wild-type and Atg7-cKO mice.

Zeroing in on one that was far more abundant in the conditional knockouts, they found a cadre of microglia that appeared to have transitioned into a senescent state. These “pretend aged” cells expressed cyclin-dependent kinase inhibitor Cdkn1a, which causes senescence, as well as an abundance of chemokines including Ccl3, Ccl6, and Ccl9. This gene-expression profile overlapped markedly with senescent microglial clusters previously identified in old mice and in aged human brain (Dec 2018 news; Olah et al., 2018; Nov 2019 news). What’s more, microglia in these mice assumed a dystrophic morphology, projecting shorter and fewer branches. This happened without amyloid plaques or similar irritants, nearby.

DAM to SAM. In 5xFAD mice (top), plaque-adjacent microglia (green) express an abundance of the DAM marker Clec7a (red). 5xFAD mouse microglia lacking autophagy (bottom) also lack Clec7a, and the autophagy substrate p62 accumulates (bottom). [Courtesy of Choi et al., 2023]

In 5xFAD mice crossed to these conditional knockouts, microglia also did not assume a DAM state, opting instead for a senescence-associated profile. “SAMs” expressing S100a4, a marker of this senescent profile, appeared disinterested in amyloid. As a result, Aβ sprawled into diffuse plaques, which were surrounded by hyperphosphorylated tau and dystrophic neurites. This suggests that, without autophagy available to them, microglia became recalcitrant and no longer contained Aβ plaque formation, allowing the aggregates to become more of a hazard to nearby neurons.

Finally, the researchers treated the double-transgenic mice with dasatinib and quercetin, a combination therapy with proposed senolytic effects. A Phase 1/2 clinical trial of it in people who have early AD and a positive tau PET scan is slated to wrap up at the end of this year. In the mice, the treatment reduced the number of microglia that were clogged with a backlog of autophagy substrates and expressed senescence markers. In contrast, numbers of plaque-associated microglia expressing the DAM marker, Clec7a, slightly increased. As a result, Aβ plaques were rounder and more compact, with fewer dystrophic neurites surrounding them.

The findings place autophagy upstream of the microglial transition to a beneficial, disease-associated state, the authors proposed. Considering reports that autophagy declines with age while senescent cells become more numerous, the authors blame this combination for the dearth of DAM-like cells detected in human AD brains (May 2019 news). “It is conceivable that age-related reduction or gene perturbation of autophagy in microglia could result in senescence and suppression of DAM, contributing to the acceleration of Aβ-linked pathology in AD,” they wrote.

Scientists led by Seiko Ikezu at the Mayo Clinic in Jackonville, Florida, came to similar conclusions. They reported that blocking microglial endolysosomal processing—in this case, biogenesis of extracellular vesicles—clogs microglial digestion and thwarts transition into the DAM state (Apr 2023 conference news). 

That said, Choi’s findings differ from those of Diego Gomez-Nicola at the University of Southampton, U.K. His work placed microglial senescence downstream of DAM (Jun 2021 news). Still, both studies suggest that, once senescent, microglia do more harm than good.—Jessica Shugart

Comments

  1. This interesting study from Dr. Yue’s group reveals the novel autophagy-mediated regulatory mechanism of disease-associated/neurodegenerative microglia (DAM/MGnD) phenotype induction (Krasemann et al., 2017) via microglia-specific targeting of Atg7. I am intrigued that autophagosomal machinery may regulate microglial activation, because we recently observed that deletion from microglia of tumor susceptibility gene Tsg101, one of the ESCRT1 (endosomal sorting complex required for transport) molecules, downregulated DAM/MGnD induction in PS19 mice (Apr 2023 news). Interestingly, we found the Tsg101 cKO-microglia significantly suppressed phagocytic function in vitro, which could lead the suppression of DAM/MGnD in PS19 mice since phagocytosis of dead neurons is an established method for the induction of this phenotype (Krasemann et al., 2017). While the authors showed that microglial deletion of Atg7, a critical molecule for the autophagosome machinery, in 5XFAD mice downregulated DAM/MGnD signature genes, they did not examine phagocytic function in Atg7cKO microglia. We may expect similar results since phagocytosis and autophagy share molecular pathways for evolutionally conserved functions in myeloid cells (Eshraghi et al., 2021). Indeed, Atg7 activation recruits LC3, beclin, and other phagocytosis-related molecules to the phagosome membranes. Thus, it is possible that Atg7 cKO downregulates DAM/MGnD induction via suppression of microglial phagocytosis.

    The authors also suspect that senescence triggered by autophagy dysfunction could be the mechanism that explains the downregulation of DAM. However, DAM/MGnD genes, such as APOE, Cst7, and CD74, are upregulated as well as the senescence gene Cdkn1a, in Atg7cKO microglia, according to their single RNA-Seq analysis. Indeed, the Gomez-Nicola group recently reported positive correlation between microglial senescence and DAM/MGnD induction (Hu et al., 2021). Thus, the regulation of DAM/MGnD induction by senescence may be context-dependent (presence or absence of disease status), and this finding needs more validation with senescence-specific targeting approaches.

    References:

    Krasemann S, Madore C, Cialic R, Baufeld C, Calcagno N, El Fatimy R, Beckers L, O'Loughlin E, Xu Y, Fanek Z, Greco DJ, Smith ST, Tweet G, Humulock Z, Zrzavy T, Conde-Sanroman P, Gacias M, Weng Z, Chen H, Tjon E, Mazaheri F, Hartmann K, Madi A, Ulrich JD, Glatzel M, Worthmann A, Heeren J, Budnik B, Lemere C, Ikezu T, Heppner FL, Litvak V, Holtzman DM, Lassmann H, Weiner HL, Ochando J, Haass C, Butovsky O. The TREM2-APOE Pathway Drives the Transcriptional Phenotype of Dysfunctional Microglia in Neurodegenerative Diseases. Immunity. 2017 Sep 19;47(3):566-581.e9. PubMed.

    Eshraghi M, Adlimoghaddam A, Mahmoodzadeh A, Sharifzad F, Yasavoli-Sharahi H, Lorzadeh S, Albensi BC, Ghavami S. Alzheimer's Disease Pathogenesis: Role of Autophagy and Mitophagy Focusing in Microglia. Int J Mol Sci. 2021 Mar 24;22(7) PubMed.

    Hu Y, Fryatt GL, Ghorbani M, Obst J, Menassa DA, Martin-Estebane M, Muntslag TA, Olmos-Alonso A, Guerrero-Carrasco M, Thomas D, Cragg MS, Gomez-Nicola D. Replicative senescence dictates the emergence of disease-associated microglia and contributes to Aβ pathology. Cell Rep. 2021 Jun 8;35(10):109228. PubMed.

    View all comments by Seiko Ikezu

  2. In this paper, inhibition of microglial autophagy induced senescence, suppressed disease-associated microglia, impaired engagement with Aβ plaques, and aggravated neuropathology. The senolytic drugs dasatinib and quercetin removed senescent microglia, restored DAMs and homeostasis of Aβ plaques, and reduced neuropathology. It is interesting to link microglial autophagy and senescence with Aβ plaque regulation and AD pathogenesis.

    Previous research has interpreted DAMs as a toxic contributor to AD, which has led to therapeutic strategies aimed at calming DAMs. However, this paper provides new insight into the possibility that DAMs are actually functioning microglia that reduce AD pathology, and that senescent and nonfunctioning microglia are the ones that aggravate AD pathology. This may suggest a new therapeutic strategy of enhancing young DAMs and removing senescent microglia.

    However, it is not yet clear whether DAMs are beneficial or harmful. It is possible that their role may differ depending on the stage and environment. Microglia may change from normal active microglia to hyper-immune and inflammatory, and then to senescent and degenerating microglia, over the long duration of aging and AD. More research is needed to fully understand the role of microglia in AD and to develop effective therapeutic strategies.

    Overall, this study provides valuable new insights into the role of microglia in AD and suggests that senolytic drugs may be a potential treatment for the disease. However, more research is needed to confirm these findings and to develop effective therapeutic strategies.

    View all comments by Seung-Yong Yoon

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References

News Citations

  1. Death of the Neatnik: Neurons Perish When Trash Clutters Their Space?
  2. Behold PANTHOS, a Toxic Wreath of Perinuclear Aβ That Kills Neurons
  3. Without TREM2, Microglia Run Out of Gas
  4. Can Loss of a Single Protein Domain Cause ‘Alzheimer’s’ in Mice?
  5. Hot DAM: Specific Microglia Engulf Plaques
  6. Shooting Themselves in the Foot? Microglia Block “Good” State with ApoE4
  7. Microglia Reveal Formidable Complexity, Deep Culpability in AD
  8. The Human Brain Hosts a Menagerie of Microglia
  9. When It Comes to Alzheimer’s Disease, Do Human Microglia Even Give a DAM?
  10. From Phagocytosis to Exophagy: Microglia's Digestive Tract Dissected
  11. DAMned to Death? Microglia May Proliferate to Senescence

Therapeutics Citations

  1. Dasatinib + Quercetin

Paper Citations

  1. Nixon RA, Wegiel J, Kumar A, Yu WH, Peterhoff C, Cataldo A, Cuervo AM. Extensive involvement of autophagy in Alzheimer disease: an immuno-electron microscopy study. J Neuropathol Exp Neurol. 2005 Feb;64(2):113-22. PubMed.
  2. Xu Y, Propson NE, Du S, Xiong W, Zheng H. Autophagy deficiency modulates microglial lipid homeostasis and aggravates tau pathology and spreading. Proc Natl Acad Sci U S A. 2021 Jul 6;118(27) PubMed.
  3. Olah M, Patrick E, Villani AC, Xu J, White CC, Ryan KJ, Piehowski P, Kapasi A, Nejad P, Cimpean M, Connor S, Yung CJ, Frangieh M, McHenry A, Elyaman W, Petyuk V, Schneider JA, Bennett DA, De Jager PL, Bradshaw EM. A transcriptomic atlas of aged human microglia. Nat Commun. 2018 Feb 7;9(1):539. PubMed.

Further Reading

Papers

  1. Cho MH, Cho K, Kang HJ, Jeon EY, Kim HS, Kwon HJ, Kim HM, Kim DH, Yoon SY. Autophagy in microglia degrades extracellular β-amyloid fibrils and regulates the NLRP3 inflammasome. Autophagy. 2014 Oct 1;10(10):1761-75. Epub 2014 Jul 22 PubMed.

Primary Papers

  1. Choi I, Wang M, Yoo S, Xu P, Seegobin SP, Li X, Han X, Wang Q, Peng J, Zhang B, Yue Z. Autophagy enables microglia to engage amyloid plaques and prevents microglial senescence. Nat Cell Biol. 2023 Jul;25(7):963-974. Epub 2023 May 25 PubMed.

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