Marschallinger J, Iram T, Zardeneta M, Lee SE, Lehallier B, Haney MS, Pluvinage JV, Mathur V, Hahn O, Morgens DW, Kim J, Tevini J, Felder TK, Wolinski H, Bertozzi CR, Bassik MC, Aigner L, Wyss-Coray T. Lipid-droplet-accumulating microglia represent a dysfunctional and proinflammatory state in the aging brain. Nat Neurosci. 2020 Feb;23(2):194-208. Epub 2020 Jan 20 PubMed. Correction.

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  1. Recent evidence showed that, during aging, microglia accumulate lipids within the phagosomes. Hence, it has been suggested that lipid-rich microglia would less efficiently uptake and degrade cellular debris or protein aggregates, and this may contribute to brain aging and neurodegenerative diseases (Cantuti-Castelvetri et al., 2018; Safaiyan et al., 2016). This recent work by Marschallinger and colleagues has further elucidated this phenomenon, showing that lipid-rich microglia accumulate in the hippocampi of both aging mice and senile humans.

    Using RNA sequencing, authors showed that lipid-rich microglia exhibit a different gene expression compared with microglia with low lipid content. Interestingly, most of the upregulated genes are involved in phagosomes maturation and ROS production. The authors named this specific microglial phenotype “Lipid droplet Accumulating Microglia,” or LAM. Using different assays, they demonstrated that LAM exhibit reduced phagocytic capacity and increased production of ROS and pro-inflammatory cytokines. Lastly, the authors showed that brains of progranulin-deficient mice (Gran-/-) exhibit abundant LAMs already during adulthood.

    In humans, Grn mutations have been linked to frontotemporal dementia, therefore accumulation of lipid droplets in human microglia may play an important role in the context of neurodegenerative diseases. Overall, this work highlights new compelling features of microglial aging, delineating a possible mechanism linking lipid-rich microglia and dementia. For the future, it will be interesting to determine whether certain Alzheimer’s risk alleles (such as ApoE-ε4 or Trem2 R47H) could exacerbate the LAM formation, thus accelerating brain aging and deposition of Aβ.

    References:

    Cantuti-Castelvetri L, Fitzner D, Bosch-Queralt M, Weil MT, Su M, Sen P, Ruhwedel T, Mitkovski M, Trendelenburg G, Lütjohann D, Möbius W, Simons M. Defective cholesterol clearance limits remyelination in the aged central nervous system. Science. 2018 Feb 9;359(6376):684-688. Epub 2018 Jan 4 PubMed.

    Safaiyan S, Kannaiyan N, Snaidero N, Brioschi S, Biber K, Yona S, Edinger AL, Jung S, Rossner MJ, Simons M. Age-related myelin degradation burdens the clearance function of microglia during aging. Nat Neurosci. 2016 Aug;19(8):995-8. Epub 2016 Jun 13 PubMed.

    View all comments by Simone Brioschi

  2. This exciting new study by Marschallinger et al. defines and examines the age-dependent accumulation of lipid droplets within microglia, clearly demonstrating that these lipid-accumulating microglia (LAMs) exhibit a unique transcriptional signature, pro-inflammatory phenotype, and impaired phagocytosis. Interestingly, LAM are far more prevalent in the hippocampus than other brain regions. While the cause of this hippocampal LAM enrichment remains unclear, it is interesting to speculate that this may be associated with the turnover of newborn neurons, given the role of microglia in the clearance of apoptotic cells within neurogenic niches (Fourgeaud et. al., 2016). 

    Focusing in on the hippocampus, Marschallinger and colleagues use BODIPY, which detects neutral lipids, to quantify and isolate LAM and compare them to microglia with low lipid content. They find that the number of LAM are more than fourfold higher in 20-month-old versus 3-month-old mice, and likewise using an antibody approach provide initial evidence that LAM are also increased in aged human brains. The authors then use their BODIPY FACS approach to examine the transcriptome and functions of LAM, finding significant changes in in phagosome maturation and pro-inflammatory pathways.

    While the acronym LAM was also recently introduced by Ido Amit in reference to “lipid-associated macrophages” from white adipose tissue (Jaitin et al., 2019), the LAM described in the current study differ considerably from these adipose LAM. Whereas adipose-LAM exhibit transcriptional profiles that closely mimic signatures of disease-associated microglia (DAM), the age-associated microglial-LAM described in the current study exhibit profiles that are relatively inverse to that of AD-associated DAM and appear to be TREM2-independent. As the presence of amyloid plaques and dystrophic neurites likely alters the lipids that microglia are exposed to, it will be very interesting to learn how the lipid content of DAM differs from that of adipose- and age-associated LAM and whether human DAM and LAM exhibit similar profiles to their murine counterparts.

    In this respect, we recently demonstrated considerable differences between human and murine DAM genes in a chimeric AD model, suggesting important species differences exist in these various microglial phenotypes (Hasselman et al., 2019). Interestingly, our ongoing studies have also revealed lipid accumulation and enrichment of many lipid-associated genes in human plaque-associated DAM. Given the growing list of lipid-associated AD risk genes (APOE, TREM2, Clusterin, ABCA7, SORL1, MS4As), it will be fascinating to determine how the composition of lipids that accumulate in human DAMs and LAMs are influenced by these genes and to watch how this exciting story continues to evolve.

    References:

    Fourgeaud L, Través PG, Tufail Y, Leal-Bailey H, Lew ED, Burrola PG, Callaway P, Zagórska A, Rothlin CV, Nimmerjahn A, Lemke G. TAM receptors regulate multiple features of microglial physiology. Nature. 2016 Apr 14;532(7598):240-244. Epub 2016 Apr 6 PubMed.

    Jaitin DA, Adlung L, Thaiss CA, Weiner A, Li B, Descamps H, Lundgren P, Bleriot C, Liu Z, Deczkowska A, Keren-Shaul H, David E, Zmora N, Eldar SM, Lubezky N, Shibolet O, Hill DA, Lazar MA, Colonna M, Ginhoux F, Shapiro H, Elinav E, Amit I. Lipid-Associated Macrophages Control Metabolic Homeostasis in a Trem2-Dependent Manner. Cell. 2019 Jul 25;178(3):686-698.e14. Epub 2019 Jun 27 PubMed.

    Hasselmann J, Coburn MA, England W, Figueroa Velez DX, Kiani Shabestari S, Tu CH, McQuade A, Kolahdouzan M, Echeverria K, Claes C, Nakayama T, Azevedo R, Coufal NG, Han CZ, Cummings BJ, Davtyan H, Glass CK, Healy LM, Gandhi SP, Spitale RC, Blurton-Jones M. Development of a Chimeric Model to Study and Manipulate Human Microglia In Vivo. Neuron. 2019 Sep 25;103(6):1016-1033.e10. Epub 2019 Jul 30 PubMed.

    View all comments by Christel Claes

  3. Microglia heterogeneity has just started being appreciated. Their diversity likely depends upon intrinsic properties as well as environmental cues within their niche, including specific regional localization. Overlying this diversity, microglia are also dynamic, and may switch between different states in response to an insult. Several phenotypes have been already identified in conditions such as aging or disease. However, the roles of microglia in these different states is far from complete.

    Against this backdrop, Marschallinger et al. characterized an additional functional state of microglia that seemingly reflects changes in lipid metabolism and is associated with inflammation, aging, and neurodegeneration. Interestingly, only partial overlap was found with previously described “diseased-microglia” signatures. The so-called “lipid-droplet accumulating microglia” (LAM) constitute a proportion of the microglia population that increases during aging, and seems to be present predominantly in the hippocampus rather than in other brain regions. Is there a regional specificity to the phenotype observed, e.g., does this phenotype reflect regional selectivity to aging? Using a model for frontotemporal dementia (FTD), the authors found additional LAM in the thalamus, a region particularly affected by degeneration in FTD. This suggests that a selective vulnerability might also be linked to the specific disease-context. In any case, LAM seem to concentrate in areas subjected to stress. Whether specific subtypes of microglia within these areas are particularly susceptible to metabolic stress remains to be addressed.

    Leukocytes, macrophages, and microglia containing accumulations of lipids upon inflammation have been described before. Lipid droplets have been associated with inflammation and cytokine storage, but also with homeostatic functions, such as storage of fatty acids that could support phagocytosis and proper mitochondria functioning (Nadjar et al., 2018). More broadly, a role for glial cells in maintaining lipid homeostasis has been described, with a recent paper showing that neuronal lipids are transferred to astrocytes, to counteract excitotoxic effects (Ioannou et al., 2019). Although Marschallinger et al. reported that no transcriptional changes have been detected in lipid transporter genes, suggesting that lipid droplet dynamics in microglia might differ compared with astrocytes, some changes could occur at the protein level and on a wider level, as compensatory changes of the entire lipid metabolism. It would be of great interest to assess the crosstalk between different cell types (including neurons, astrocytes, and microglia, but also potentially ependymal cells and endothelial cells lining the blood vessels) that mediate lipid metabolism in the brain.

    Through comparison of the transcriptome of LAM with non-lipid-laden microglia, the authors identified a new profile associated with lysosomal, ROS, and NO production genes, with phagocytosis and ROS generation as the highest upregulated pathways. Remarkably, LAM showed severely reduced phagocytosis accompanied by accumulation of lysosomal vesicles. Could droplet numbers increase as a consequence of impaired cell function (e.g., reduced mitochondria functioning and impaired phagocytosis) and reduced lysis? As suggested by the authors, lysosomal defects might underlie the accumulation observed. At the same time, the droplets themselves might be the cause for the altered homeostatic functioning by “sequestering” the lysosomes normally involved in phagocytosis. What is clear is that the phenotype of this cell is detrimental to properly functioning microglia.

    One critical question still to be addressed is whether the LAM transcriptional profile can be mapped onto aging and/or diseased microglia in the human brain (such as the recently described HAM signature, Srinivasan et al., 2019). Lipofuscin deposits are often found in human brains, sometimes in association with lipid droplets, however, it is not clear whether they always co-exist and might be the consequence of distinct processes (Shimabukuro et al., 2016). Lipofuscin deposits are a hallmark of a number of degenerative disorders, including age-related macular degeneration (Kauppinen et al, 2017). 

    Accumulation of lipids is not a unique feature to microglia, however, it seems to be a hallmark of brain aging. GWAS have identified lipid metabolism as an important pathway in AD, and GWAS hits, such as Trem2 and ApoE, are involved in lipid sensing and processing. As mentioned in this paper, Dr. Alzheimer himself described lipid accumulation in the cell cytoplasm of AD brains. This could be an unappreciated feature that characterizes not only AD, but also other neurodegenerative pathologies. Finally, this new field of investigation opens up new questions on the processes occurring during these disorders. For instance, could AD be considered a systemic disease, where a more broadly disrupted metabolism causes locally altered homeostasis, and consequent neurodegeneration due to loss of support from homeostatic functions in the brain?

    References:

    Nadjar A. Role of metabolic programming in the modulation of microglia phagocytosis by lipids. Prostaglandins Leukot Essent Fatty Acids. 2018 Aug;135:63-73. Epub 2018 Jul 18 PubMed.

    Ioannou MS, Jackson J, Sheu SH, Chang CL, Weigel AV, Liu H, Pasolli HA, Xu CS, Pang S, Matthies D, Hess HF, Lippincott-Schwartz J, Liu Z. Neuron-Astrocyte Metabolic Coupling Protects against Activity-Induced Fatty Acid Toxicity. Cell. 2019 May 30;177(6):1522-1535.e14. Epub 2019 May 23 PubMed.

    Srinivasan K, Friedman BA, Etxeberria A, Huntley MA, van der Brug MP, Foreman O, Paw JS, Modrusan Z, Beach TG, Serrano GE, Hansen DV. Alzheimer's Patient Microglia Exhibit Enhanced Aging and Unique Transcriptional Activation. Cell Rep. 2020 Jun 30;31(13):107843. PubMed.

    Shimabukuro MK, Langhi LG, Cordeiro I, Brito JM, Batista CM, Mattson MP, Mello Coelho Vd. Lipid-laden cells differentially distributed in the aging brain are functionally active and correspond to distinct phenotypes. Sci Rep. 2016 Mar 31;6:23795. PubMed.

    View all comments by Lorenza Magno

  4. This is an interesting study that reminds me of Alois Alzheimer’s drawing of Nile red staining, which reveals intracellular lipid droplets (LD) in cells (potentially microglia) around plaques in AD patient brain. Here, Marschallinger and colleagues identified LD-accumulating microglia in aged brain, which led to a defect in phagocytosis, high levels of reactive oxygen species (ROS), and pro-inflammatory phenotypes.

    DAM, HAM, and LAM: stage-specific microglia captured from young to old brains?
    The authors separated cells containing high vs. low levels of LDs. High-LD microglia (50 percent of those in the aged brain), called LAM, showed unchanged expression of APOE, which is one of the key markers for disease-associated microglia (DAM) polarization, but expression changes in many other genes that were the opposite of that seem in DAM. However, the other half of the aged brain microglia, which are low-LD, may still have a DAM-like signature. The authors’ findings suggest there is an LD-dependent divergence of beneficial and detrimental microglia, which co-exist in aged brain. The relative proportions of these cells probably changes during the time course of aging and are affected by genetic mutations and disease risk factors. Further comparison of the overlap between LAM and human Alzheimer’s microglia (HAM), which represents an enhanced aging phenotype (Galatro et al., 2017; Srinivasan et al., 2019), may reveal age-associated gene-expression changes shared in both mouse and human microglia. Surprisingly, Triacsin C easily reverts the LAM phenotype to normal, but we may need to investigate further how blocking de novo synthesis of lipids affects cellular lipid homeostasis and if it would have potential adverse effects if used as a therapeutic intervention.

    Differences between lipid-accumulating microglia and LAM (lipid droplet accumulating microglia)
    We need to use the acronym LAM carefully. This study is focused on a cellular organelle, the lipid droplet, but not the entire complement of cellular lipids. There are studies that show microglia accumulate cholesterol after excessive uptake of extracellular lipids or if they express the AD genetic risk factor APOE 4 (Cantuti-Castelvetri et al., 2018; TCW et al., 2019). These lipid-accumulating microglia—i.e., not LAM—possess mainly free (unesterified) cholesterol, which is converted from extracellular lipids or generated through biosynthesis. In LAM, Marschallinger and colleagues found almost no cholesteryl esters in LD of aged microglia and aged hippocampal mouse brain, and their data also suggested that demyelination does not contribute to LD in LAM. Consistent with unaffected levels of cholesteryl ester by APOE4, as measured by gas chromatography-mass spectrometry (TCW et al., 2019), the authors also observed that APOE and other lipid transporters were not significantly regulated in LAM. However, to address cholesterol accumulation in aged microglia, total cholesterol as well as intracellular free cholesterol should be considered.

    Regional/cell-type-specific differences in lipid composition of LD
    Lipidomic analysis reported in this preprint showed glycerolipids and phospholipids are the major content of LAM. It is also interesting to see the different composition of brain vs. peripheral cells (liver), for example sphingomyelin, which forms a partner with cholesterol in plasma membranes that control cholesterol synthesis, is high in liver cells but not in brain microglia. We should further explore microglia specific lipid regulation.

    What’s first, inflammation or lipid accumulation? Whichever comes first, LAM do not phagocytose!
    In transcriptome, authors found that lipopolysaccharide (LPS) is the most significant upstream regulator in high LD microglia. Further in vitro validation showed that LPS treatment of BV2 cells and hippocampal microglia in young mice increases lipid droplets, indicating inflammation-induced LD formation. Many other factors can induce LD formation, however, the important finding is that LAM in aged brain do not phagocytose, displaying a unique transcriptomic signature as well as a detrimental phenotype.

    References:

    Galatro TF, Holtman IR, Lerario AM, Vainchtein ID, Brouwer N, Sola PR, Veras MM, Pereira TF, Leite RE, Möller T, Wes PD, Sogayar MC, Laman JD, den Dunnen W, Pasqualucci CA, Oba-Shinjo SM, Boddeke EW, Marie SK, Eggen BJ. Transcriptomic analysis of purified human cortical microglia reveals age-associated changes. Nat Neurosci. 2017 Aug;20(8):1162-1171. Epub 2017 Jul 3 PubMed.

    Srinivasan K, Friedman BA, Etxeberria A, Huntley MA, van der Brug MP, Foreman O, Paw JS, Modrusan Z, Beach TG, Serrano GE, Hansen DV. Alzheimer's Patient Microglia Exhibit Enhanced Aging and Unique Transcriptional Activation. Cell Rep. 2020 Jun 30;31(13):107843. PubMed.

    Cantuti-Castelvetri L, Fitzner D, Bosch-Queralt M, Weil MT, Su M, Sen P, Ruhwedel T, Mitkovski M, Trendelenburg G, Lütjohann D, Möbius W, Simons M. Defective cholesterol clearance limits remyelination in the aged central nervous system. Science. 2018 Feb 9;359(6376):684-688. Epub 2018 Jan 4 PubMed.

    Tcw J, Qian L, Pipalia NH, Chao MJ, Liang SA, Shi Y, Jain BR, Bertelsen SE, Kapoor M, Marcora E, Sikora E, Andrews EJ, Martini AC, Karch CM, Head E, Holtzman DM, Zhang B, Wang M, Maxfield FR, Poon WW, Goate AM. Cholesterol and matrisome pathways dysregulated in astrocytes and microglia. Cell. 2022 Jun 23;185(13):2213-2233.e25. PubMed. BioRxiv.

    View all comments by Julia TCW

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