Ridding Microglia of a MicroRNA Compacts Amyloid, Protects Synapses

Relinquishing microglia of a pesky 22-nucleotide microRNA freed the cells to respond more vigorously to amyloidosis, and to protect nearby neurons. That was the upshot of a study published in Nature Neuroscience on June 8, which was spurred by previous findings implicating this particular microRNA—miR155—in microglial function. When the scientists, led by Oleg Butovsky of Brigham and Women’s Hospital in Boston and Tsuneya Ikezu of the Mayo Clinic in Jacksonville, Florida, conditionally deleted miR-155 from microglia in APP/PS1 mice, the cells heightened their responses to IFN-γ. This bolstered phagocytosis, boosted containment of Aβ plaques, and ultimately protected synapses and spared memory loss. Notably, these benefits depended upon the interferon, a cytokine produced primarily by T cells. The findings underscore the complexity—and rich opportunities for therapeutic targets—embedded within the multi-pronged inflammatory response to amyloidosis.

  • Sans microRNA-155, microglia respond better to interferon-γ.
  • They more readily compact plaques.
  • Synapses were spared, and memory preserved, in miR-155 knockouts.

Despite their teeny stature, microRNAs pack a gene expression punch, dousing translation of myriad target genes. Butovsky’s group had previously pegged miR-155 as part of a  neurodegenerative microglial (MgND) signature, or what others have called a disease associated (DAM) signature, based on mouse models of amyloidosis and ALS (Sep 2017 news, Feb 2015 news). Knocking out the microRNA from an ALS model locked microglia in a homeostatic state, which proved beneficial in that context (Butovsky et al., 2015). For the new study, co-first authors Zhuoran Yin and Shawn Herron, and their colleagues, used an inducible expression system to conditionally knock out miR-155 from microglia in six-week-old APP/PS1 mice, then analyzed their microglia at four months of age. What they found surprised them, Ikezu said. Instead of being locked in a homeostatic state as in the global miR-155 knockout, microglia in the conditional KO had morphed into a responsive state. They revved up expression of multiple MGnD genes, including ApoE, Axl, Cst7, and Clec7a, as well as a host of interferon response genes. Many cell types throughout the body express miR-155, including immune cells that likely influence microglial states within the brain, and this might explain the discrepancies between the global and conditional knockouts, Ikezu speculated (reviewed in Mahesh and Biswas, 2019).

A finer-grained inspection of these transcriptional effects using single-cell RNA sequencing of brain tissue from APP/PS1 mice with or without microglial miR-155 revealed three major microglial transcriptional subtypes, based on their relative expression of homeostatic and MGnD signature genes: homeostatic, “pre-MGnD,” and MGnD. Looking closer at the pre-MGnD cells, the researchers observed both “early,” and “late” transcriptomes, based on a pseudo timeline between homeostatic and MGnD signatures. In the early phase, microglia retained expression of many homeostatic genes, while ramping up expression of a suite of IFN-response genes, such as Stat1, Isg15, Ifi204, Irf7, Ifit1 and Ifit3. In the late stage, microglia quelched homeostatic genes, continued to express IFN-response genes, and started expressing MGnD genes, including Clec7a, Axl, and ApoE. Finally, MGnD microglia downregulated both homeostatic and IFN-response genes, expressing the full suite of MGnD/DAM signature genes. Ablation of miR-155 led to the proliferation of both early and late pre-MGnD clusters in APP/PS1 mice, while reducing the relative proportion of homeostatic or mature MGnD cells.

In short, the findings suggested that removal of miR-155 shifts microglial into an interferon-responsive state. But how? Yin, Herron, and colleagues found a plausible mechanism, reporting that miR-155 suppressed expression of Stat1, a critical downstream mediator of IFN-γ receptor signaling. Removing this microRNA rendered the cells exquisitely responsive to IFN-γ.

Microglia Unleashed. Relative to microglia in APP/PS1 controls (top panels), microglia lacking miR-155 (bottom panels) internalized more Aβ (red, left), and more readily gathered around plaques while maintaining expression of TMEM119 (green, middle). [Image courtesy of Yin et al., Nature Neuroscience, 2023

How did this unfettered IFN-γ response affect microglial function? For one, it stoked their appetite for apoptotic neurons injected into the brain. Notably, this boost was not observed in mice treated with an IFN-γ blocking antibody. The conditional knockouts also had a slightly lower A burden compared to APP/PS1 controls at four months of age, and the plaques they did have were more spherical and surrounded by fewer dystrophic neurites (image above). This suggested that without miR-155, microglia were more proficient at compacting plaques, thus lessening damage to nearby neurons. In line with this, the conditional knockouts lost fewer synapses, and performed like wild-type mice on memory tests. Curiously, a previous study led by Michelle Ehrlich and Joel Dudley at the Icahn School of Medicine at Mount Sinai, New York, reported that full, constitutive knockout of miR-155 exacerbated amyloid accumulation in APP/PS1 mice, yet still protected synapses and memory, again suggesting that other cells besides microglia may rely on this micro RNA for controlling amyloid (Readhead et al., 2020).

In all, the findings indicate that at least in the APP/PS1 mouse model, miR-155 holds microglia back from responding too vigorously to IFN-γ. When this inhibition is relieved, IFN-γ rouses the cells into a phagocytic, plaque-compacting state, which attenuates neuronal damage.

In contrast, scientists led by Gwenn Garden of the University of North Carolina at Chapel Hill, found a dark side to the conditional miR-155 KO/APPPS1 model. Yes, microglia contained amyloid, but they also noshed on synapses, triggered neuronal hyperexcitability, and ramped up deadly seizures in the mice (Alois et al., 2023). The authors reported that removal of miR-155 upped microglial appetite for excitatory synapses, which, counterintuitively, can lead to a synaptic imbalance that sparks epilepsy. Garden told Alzforum that the APP/PS1 mice are prone to seizures, and that differences in background strains of mice used in the two studies may have influenced this electrical imbalance. Ikezu agreed, noting that while they did not observe the same increase in overt seizures among their conditional knockouts, they did not perform electrophysiological studies, as Garden did, to look for changes in neuronal activity.

Butovsky and Ikezu think miR-155 could be a potential therapeutic target for AD, particularly since this microRNA has been found to be elevated in the AD brain (Sierksma et al., 2018). Yet, herein lies another complexity. While the researchers found that miR-155 is predominantly expressed by microglia in the mouse brain, it also appears to be expressed by neurons and other cell types in the human brain, suggesting it may have many different effects across cell types. Because microRNAs are difficult to analyze via single-cell RNA sequencing, more work is needed to nail down the expression patterns of miR-155 in the human brain, Ikezu said.

Evgenia Salta of the Netherlands Institute for Neuroscience noted that even within a single cell type, each microRNA exerts its influence over hundreds of target genes, casting the RNA snippets as promising multi-targeted approaches to therapy (comment below). “However, this exact advantage is the same that may—on the flip side—jeopardize microRNA therapeutic applications,” she noted, because the many genes regulated by each microRNA may make them unsuitable for selective therapeutic targeting. (Walgrave et al., 2021).

David Holtzman of Washington University in St. Louis noted that while removal of miR-155 from microglia appeared to benefit mice in the early stages of amyloidosis, the ablation could have different effects in people, depending on disease stage (comment below). “When other changes begin to occur within the AD brain, such as tau pathology that is linked with cognitive decline and neurodegeneration, as well as a different inflammatory environment, the microglial state induced by mIR-155 or IFN-γ may not have the same effects that are associated with neuroprotection as shown here,” he wrote. “Work in other model systems that develop neurodegeneration, such as occurs with tau pathology, may be helpful to sort out when to try to target these pathways during different stages of AD.”—Jessica Shugart

Comments

  1. This new manuscript from Yin and colleagues adds important information to our understanding of how miR-155 impacts microglia gene expression in the context of an AD model. It also confirms our previously reported findings that microglia-specific conditional knockout of miR-155 yields reduced amyloid plaque burden in APP/PS1 mice. These studies differ in that the impact of microglia-specific deletion of miR155 on seizures and mortality that we recently reported was not addressed here (Aloi et al., 2023). It is unclear if these negative effects of the same genetic manipulation were not observed, or just not reported.

    The APP/PS1 model is well known to exhibit seizures and seizure-related mortality, but background strain could influence the rates at which this phenotype is observed. The modest, but statistically significant improvement on a single behavioral assay reported in Yin et al., should be taken in context. The assay may have been performed in a subset of animals with heightened resilience to seizures or to the enhanced synaptic pruning by miR-155 deficient microglia that we observed.

    View all comments by Gwenn A. Garden

  2. I agree that the ablation of miR-155 in microglia promotes their transition into an IFN-γ responsive state in a mouse model of amyloidosis. I also agree that the data show that mIR-155 ablation in microglia leads to the cells being more adept at phagocytosis, and at containment of Aβ plaques. The effect of plaque load overall was small, but there was reduced peri-plaque neuritic dystrophy and less synaptic loss surrounding plaques. APP/PS1 mice without microglial mIR-155 performed better on memory tests, and lost fewer synapses. Notably, these improvements were decreased when mIR-155 was knocked out of microglia. They did not test whether IFN-γ blockade in APP/PS1 mice affected behavior.

    The findings are clear that miR-155 KO in microglia results in a more MgND/DAM state of gene expression and in the presence of amyloid, there is greater plaque compaction and decreased plaque associated dystrophic neurites and peri-plaque synaptic loss. It is likely that IFN-γ signaling causes similar effects as miR-155 KO of microglia in this mouse model. The work appears very well done and suggests a beneficial role of either knocking down miR-155 in microglia of stimulation of microglial IFN-γ signaling in the early phase of amyloid deposition.

    From the perspective of the translational implications of these findings, the APP/PS1 mice are a useful model of Aβ amyloidosis. The amyloid deposition that occurs in these, and similar models, develops no major neuronal and synaptic loss, and the major damage that occurs is immediately surrounding amyloid plaques. The brain changes in these mice mimic the ~20 year period called “preclinical” AD in humans, when amyloid is accumulating, cognition is normal, and there is no neurodegeneration (loss of neurons and brain volume). The kind of microglial response that occurs when miR-155 is knocked out (more MgND-like with increased IFN signature) in this “stage” of disease appears protective by resulting in more contained plaques and less associated plaque toxicity. A treatment that resulted in similar effects in humans would likely be useful during the “preclinical” phase of AD. However, when other changes begin to occur within the AD brain such as tau pathology, which is linked with cognitive decline and neurodegeneration, as well as a different inflammatory environment, the microglial state induced by miR-155 or IFN-γ may not have the same effects that are associated with neuroprotection as shown here. 

    Work in other model systems that develop neurodegeneration, such as occurs with tau pathology, may be helpful to sort out when to try to target these pathways during different stages of AD.

    View all comments by David Holtzman

  3. Disentangling the complexity of microRNA regulation in the brain is challenging. Deconvoluting the significance of microglial priming in physiology and disease is strenuous. Trying to concomitantly probe both is commendable.

    Yin et al. show that microglial deletion of miR-155 enhances induction of the MGnD microglial state, aka disease-associated microglia (DAM), via the IFN-γ pathway, which further attenuates amyloid, neuritic, and synaptic pathology, and improves cognition in AD mice. These observations provide the foundation for further exploring miR-155 as a therapeutic target in AD, as has already been proposed for other microRNAs (Walgrave et al., 2023). Our increasing understanding of both microRNA and AD mechanistic complexity puts microRNAs forward as promising multi-targeting approaches to therapy. However, this exact advantage is the same that—on the flip side—may jeopardize the safety of microRNA therapeutic applications: Do microRNAs also exert non-disease-relevant, putatively toxic effects in a given context (as it was recently shown for miR-155, Aloi et al., 2023), and could the many microRNA targets prove to be “too many” for clinical use (Walgrave et al., 2021)? 

    These questions, among others, are inevitably at the core of the study by Yin et al.

    Previous work by the Butovsky group has demonstrated a link between TREM2-APOE signaling, microglial transition to the MGnD state, and miR-155 (Krasemann et al., 2017). This microglial priming was associated (as opposed to the current findings) with the loss of protective MGnD function and the active induction of neurodegenerative phenotypes. Hence, the results of the recent Yin et al. study should be viewed through the prism of a bi- (or even multi-) modal, and disease-stage-specific effect of TREM2 on microglia pathophysiology. Interestingly, microglial deletion of miR-155 in wild-type mice resulted in the suppression of homeostatic microglial gene expression, further suggesting that the interplay between miR-155 and microglial priming is likely highly complex and would, therefore, require systematic assessment in a preclinical setting.

    The authors propose miR-155 depletion as a therapeutic strategy in AD. This is a valid approach given that increased miR-155 levels were observed in their APP/PS1 AD mouse model. Yet, this is not a consistent observation in the human AD brain (Sierksma et al., 2018; Lau et al., 2013). Further work with more and larger patient cohorts would be required to strengthen the translational relevance of the findings by Yin et al., especially given that systemic miR-155 deletion was previously shown to accelerate Aβ deposition in APP/PS1 mice (Readhead et al., 2018). 

    Another critical point, also raised by the authors, is the specificity of their genetic approach for microglial targeting. The Cx3cr1-Cre mouse line does not express Cre solely in microglia, but also in other macrophages, even if that remained below the detection or resolution threshold of the present study. Also relevant to this point, the specificity of miR-155 expression for microglia is not entirely clear in the literature: miR-155 has been previously also identified in neurons in the human brain (Sierksma et al., 2018), while prior work by corresponding author Oleg Butovsky, Howard Weiner and colleagues (Butovsky et al., 2014) showed that miR-155 is expressed at similar levels in adult mouse microglia, astrocytes, oligodendrocytes and primary embryonic cortical neuronal cultures. Hence, in terms of therapeutic relevance, it may also be valuable to address non-microglial (cell autonomous or non-cell autonomous) effects of miR-155 in the brain.

    Even though several questions remain unanswered, one should view all the challenges showcased in the work of Yin et al. as the way to move forward in our research on critically assessing the therapeutic potential of microRNAs in AD: microRNA biology is complex and context-dependent, directly and indirectly affecting a broad range of large molecular target networks (going beyond single, direct-target molecules operating in only one cellular population) in often intricate feed-forward and feedback regulatory loops. Microglial activation and its role in AD pathology and progression are similarly convoluted. Why then, should our approach to understanding the mechanisms and therapeutic targeting of miRNAs in AD be a simple one?

    References:

    Walgrave H, Penning A, Tosoni G, Snoeck S, Davie K, Davis E, Wolfs L, Sierksma A, Mars M, Bu T, Thrupp N, Zhou L, Moechars D, Mancuso R, Fiers M, Howden AJ, De Strooper B, Salta E. microRNA-132 regulates gene expression programs involved in microglial homeostasis. iScience. 2023 Jun 16;26(6):106829. Epub 2023 May 6 PubMed.

    Aloi MS, Prater KE, Sánchez RE, Beck A, Pathan JL, Davidson S, Wilson A, Keene CD, de la Iglesia H, Jayadev S, Garden GA. Microglia specific deletion of miR-155 in Alzheimer's disease mouse models reduces amyloid-β pathology but causes hyperexcitability and seizures. J Neuroinflammation. 2023 Mar 7;20(1):60. PubMed.

    Walgrave H, Zhou L, De Strooper B, Salta E. The promise of microRNA-based therapies in Alzheimer's disease: challenges and perspectives. Mol Neurodegener. 2021 Nov 6;16(1):76. PubMed.

    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.

    Sierksma A, Lu A, Salta E, Vanden Eynden E, Callaerts-Vegh Z, D'Hooge R, Blum D, Buée L, Fiers M, De Strooper B. Deregulation of neuronal miRNAs induced by amyloid-β or TAU pathology. Mol Neurodegener. 2018 Oct 12;13(1):54. PubMed.

    Lau P, Bossers K, Janky R, Salta E, Frigerio CS, Barbash S, Rothman R, Sierksma AS, Thathiah A, Greenberg D, Papadopoulou AS, Achsel T, Ayoubi T, Soreq H, Verhaagen J, Swaab DF, Aerts S, De Strooper B. Alteration of the microRNA network during the progression of Alzheimer's disease. EMBO Mol Med. 2013 Oct;5(10):1613-34. Epub 2013 Sep 9 PubMed.

    Readhead B, Haure-Mirande JV, Funk CC, Richards MA, Shannon P, Haroutunian V, Sano M, Liang WS, Beckmann ND, Price ND, Reiman EM, Schadt EE, Ehrlich ME, Gandy S, Dudley JT. Multiscale Analysis of Independent Alzheimer's Cohorts Finds Disruption of Molecular, Genetic, and Clinical Networks by Human Herpesvirus. Neuron. 2018 Jul 11;99(1):64-82.e7. Epub 2018 Jun 21 PubMed.

    Butovsky O, Jedrychowski MP, Moore CS, Cialic R, Lanser AJ, Gabriely G, Koeglsperger T, Dake B, Wu PM, Doykan CE, Fanek Z, Liu L, Chen Z, Rothstein JD, Ransohoff RM, Gygi SP, Antel JP, Weiner HL. Identification of a unique TGF-β-dependent molecular and functional signature in microglia. Nat Neurosci. 2014 Jan;17(1):131-43. Epub 2013 Dec 8 PubMed.

    View all comments by Evgenia Salta

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References

News Citations

  1. ApoE and Trem2 Flip a Microglial Switch in Neurodegenerative Disease
  2. Microglia in Disease: Innocent Bystanders, or Agents of Destruction?

Paper Citations

  1. Butovsky O, Jedrychowski MP, Cialic R, Krasemann S, Murugaiyan G, Fanek Z, Greco DJ, Wu PM, Doykan CE, Kiner O, Lawson RJ, Frosch MP, Pochet N, Fatimy RE, Krichevsky AM, Gygi SP, Lassmann H, Berry J, Cudkowicz ME, Weiner HL. Targeting miR-155 restores abnormal microglia and attenuates disease in SOD1 mice. Ann Neurol. 2015 Jan;77(1):75-99. Epub 2014 Nov 27 PubMed.
  2. Mahesh G, Biswas R. MicroRNA-155: A Master Regulator of Inflammation. J Interferon Cytokine Res. 2019 Jun;39(6):321-330. Epub 2019 Mar 20 PubMed.
  3. Readhead B, Haure-Mirande JV, Mastroeni D, Audrain M, Fanutza T, Kim SH, Blitzer RD, Gandy S, Dudley JT, Ehrlich ME. miR155 regulation of behavior, neuropathology, and cortical transcriptomics in Alzheimer's disease. Acta Neuropathol. 2020 Sep;140(3):295-315. Epub 2020 Jul 14 PubMed.
  4. Aloi MS, Prater KE, Sánchez RE, Beck A, Pathan JL, Davidson S, Wilson A, Keene CD, de la Iglesia H, Jayadev S, Garden GA. Microglia specific deletion of miR-155 in Alzheimer's disease mouse models reduces amyloid-β pathology but causes hyperexcitability and seizures. J Neuroinflammation. 2023 Mar 7;20(1):60. PubMed.
  5. Sierksma A, Lu A, Salta E, Vanden Eynden E, Callaerts-Vegh Z, D'Hooge R, Blum D, Buée L, Fiers M, De Strooper B. Deregulation of neuronal miRNAs induced by amyloid-β or TAU pathology. Mol Neurodegener. 2018 Oct 12;13(1):54. PubMed.
  6. Walgrave H, Zhou L, De Strooper B, Salta E. The promise of microRNA-based therapies in Alzheimer's disease: challenges and perspectives. Mol Neurodegener. 2021 Nov 6;16(1):76. PubMed.

Further Reading

Papers

  1. Shi H, Yin Z, Koronyo Y, Fuchs DT, Sheyn J, Davis MR, Wilson JW, Margeta MA, Pitts KM, Herron S, Ikezu S, Ikezu T, Graham SL, Gupta VK, Black KL, Mirzaei M, Butovsky O, Koronyo-Hamaoui M. Regulating microglial miR-155 transcriptional phenotype alleviates Alzheimer's-induced retinal vasculopathy by limiting Clec7a/Galectin-3+ neurodegenerative microglia. Acta Neuropathol Commun. 2022 Sep 8;10(1):136. PubMed.

Primary Papers

  1. Yin Z, Herron S, Silveira S, Kleemann K, Gauthier C, Mallah D, Cheng Y, Margeta MA, Pitts KM, Barry JL, Subramanian A, Shorey H, Brandao W, Durao A, Delpech JC, Madore C, Jedrychowski M, Ajay AK, Murugaiyan G, Hersh SW, Ikezu S, Ikezu T, Butovsky O. Identification of a protective microglial state mediated by miR-155 and interferon-γ signaling in a mouse model of Alzheimer's disease. Nat Neurosci. 2023 Jul;26(7):1196-1207. Epub 2023 Jun 8 PubMed.

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