Triglyceride metabolism controls inflammation and APOE4-associated disease states in microglia

Background

Lipid droplets are organelles that essentially store neutral lipids in intracellular compartments, mostly triacylglycerols and sterol esters(1,2). Lipid droplets take part in several cellular functions other than energy storage, such as mounting a response to different types of cellular stress (especially lipotoxic stress due to lipid peroxidation), starvation, oxidation and inflammation(2). Exacerbated accumulation of lipid droplets and their metabolic dysregulation are often linked with a variety of diseases(1). 

Apolipoprotein E (APOE) is present in different lipoproteins and mediates their distribution through interactions with plasma membrane receptors(3,4), regulating lipid plasma levels and their transport(3,4). There are three different isoforms, namely APOE2, APOE3 and APOE4. APOE4 is considered one of the main genetic risk factors for sporadic Alzheimer’s disease (AD), expressed in nearly half of AD patients. This isoform has been linked to cognitive impairment, mitochondrial dysfunction(4), neurotoxicity and overall lipid metabolic alterations in different cells, such as neurons and glial cells(3). Because of that, APOE4 is becoming a target to prevent or treat AD(5). Recent papers have demonstrated that APOE4 promotes microglial lipid droplets accumulation(6,7). 

Microglia are considered to be the resident immune cells of the brain(8). Being essentially macrophages, microglia are responsible to protect the nervous system against multiple injuries and diseases(8,9). When activated, microglia change their phenotype depending on the stimuli, which include pathogens, amyloid-β peptides, damage-associated molecular patterns and aging(8,10). Frequently, activated microglial cells display pro-inflammatory characteristics, including the production and secretion of cytokines, increased chemokines and enhanced phagocytosis(8,9). Like other macrophages, microglia decrease oxidative metabolism in response to inflammation, which reduces fatty acid oxidation, resulting in the accumulation of intracellular lipid droplets, indicating dysfunctional, reactive and aged microglia(6,7,10). However, the relation between lipid droplets and how they interfere and relate with microglial activation to internal or external stimuli are not well understood yet, which are the main questions authors try to answer in this preprint. 

Key findings 

LPS-activated microglia display neutral lipid accumulation 

Microglia derived from iPSCs carrying the APOE3 genotype exhibited heightened secretion of proinflammatory cytokines and chemokines, increased expression of genes associated with immune activation, enhanced phagocytic activity and morphological changes following LPS stimulation. Transcriptomic analysis revealed an upregulation of genes involved in lipid synthesis and downregulation of genes associated with lipid catabolism upon LPS activation. These transcriptional changes correlated with the intracellular increase of lipid droplets within the microglia. 

Triglyceride biosynthesis is necessary for LPS-mediated activation of microglia 

To investigate the functional implications of lipid accumulation, the authors pharmacologically inhibited DGAT1 and DGAT2 (involved in fatty acid esterification). Inhibition of these enzymes reduced triglyceride accumulation and altered the transcriptional response to LPS, affecting genes involved in both lipid metabolism and inflammation. DGAT inhibition also decreased NF-κB nuclear translocation and altered chromatin accessibility, indicating downstream effects on inflammatory gene expression. Furthermore, inhibition of triglyceride biosynthesis attenuated microglial amyloid-β phagocytosis in response to the LPS stimulus, suggesting a regulatory role of lipid metabolism in microglial activation and function. 

Triglyceride catabolism is necessary for LPS-mediated activation of microglia 

Using inhibitors of adipose triglyceride lipase (ATGL) and the phospholipase DDHD2, enzymes involved in triglyceride and phospholipid catabolism, the authors evaluated whether the presence of triglyceride-rich lipid droplets could itself activate microglia. They found that these inhibitors increased lipid droplet accumulation in human iPSC-derived microglia but did not induce cytokine secretion in inactivated cells. However, secretion of multiple cytokines was strongly reduced upon LPS stimulation when ATGL, but not DDHD2, was blocked. This strongly indicates that phospholipid metabolism does not play a role in the microglial response to external activation signals. Moreover, ATGL inhibition also impaired the phagocytosis of amyloid-β following LPS stimulation. 

Modulating triglyceride biosynthesis controls the immune state of APOE4 microglia

APOE4 microglia were found to accumulate more lipid droplets than APOE3 microglia even without external LPS stimuli, and both showed increased lipid droplets upon activation. Using DGAT1 and DGAT2 inhibitors, the authors observed that, in unstimulated cells, APOE4 microglia were more affected than APOE3 microglia. DGAT inhibition in APOE4 microglia downregulated genes related to lipid biosynthesis and immune signaling, reducing cytokine expression and secretion, independently of chromatin accessibility and structural changes. Furthermore, DGAT inhibition in both APOE3 and APOE4 microglia normalized disease-associated gene expression patterns.

Graphical abstract

Figure 1: Graphical summary of the Stephenson et al. preprint showing that lipid metabolism participates in phenotypical changes induced by LPS or APOE4 on human iPSC-derived microglia. Image drawn with Biorender.

Why I think this preprint is important

The authors show in an elegant, well-structured and easy-to-understand way that triglyceride metabolism is directly related to microglial activation. The transcriptional and functional alterations induced by LPS stimuli in different human iPSC-derived microglia with different genetic backgrounds appear to be highly dependent on triglyceride biosynthesis and degradation, especially in the APOE4 genotype. These alterations were blocked by inhibitors of enzymes related to fatty acid esterification (DGAT1 and DGAT2) and triglyceride catabolism (ATGL), suggesting that modulation of these pathways could be potentially interesting as a treatment to control neuroinflammation. Through this work, the researchers add more data that strengthen the association between lipid metabolism and inflammation. The authors also open up new possibilities with regard to AD treatment through controlling triglyceride metabolism. Even with its limitations, metabolic pathways modulation is a promising intervention for a myriad of diseases and should be studied in the next years. 

Questions and suggestions

Q1: At the end of the first paragraph of the “LPS-activated microglia display neutral lipid accumulation” section there is a typographical error. It now says “CX3XR1” instead of “CX3CR1”. 

Q2: The authors could consider standardizing their sample size data presentation. Normally different biological cultures count as different samples and different wells or cells just as experimental replicates. Standardizing this aspect of the paper would increase overall comparability and understanding of the results. 

Q3: The authors could mention the techniques used to perform the experiments in figure legends and in the results section. Although the methods were described in the ‘Materials and Methods’ section, I think it would be beneficial for the reader to also get this information while the results are presented. 

Q4: The authors could perhaps consider revising some of the data presentation throughout the manuscript. For example, would it be possible to include data from the vehicle, LPS, and LPS + inhibitor groups in the same figures/graphs? Analyzing figures S2 C, J, and L together would for instance provide a clearer picture of the specific effects of LPS activation and DGAT/ATGL inhibition on target genes, helping to evaluate whether these treatments attenuate or completely reverse the alterations caused by LPS. This type of data display would offer a broader understanding of the effects of DGAT/ATGL inhibition. 

Q5: Did the authors measure parameters like size, diameter or area of the lipid droplets? According to Benador et al., 2019(11), lipid droplets size and area are related to functional specialization of nearby mitochondria and could suggest different lipid droplet roles. Additionally, size and morphology alterations are related to differences in lipid metabolism in hepatocytes, which determine whether lipid droplets are performing lipolysis or lipophagy(12). One suggestion would be to stain the cells with BODIPY rather than LipidSPOT. This different method would allow the acquisition of confocal lipid droplets images, which is a more effective method to determine lipid droplets size. 

Q6: It would be good to specify the conditions in which the cells were harvested (medium, temperature, CO2, etc) in the ‘Materials and Methods’ section. 

Q7: Did the authors perform a viability assay on cells stimulated with LPS at 5µgmL? The concentration of LPS used as an external stimulus to promote microglial activation is relatively high and could possibly be inducing cellular death. Additionally, could the authors please explain or reference why they chose this specific stimulation protocol (concentration and duration)? One might argue that microglial cells can be stimulated with lower LPS concentrations and using higher ones might mitigate the fact that vehicle treated cells could already be activated. Either way, I think it would be worth mentioning that inhibitors were able to reverse or attenuate the effects of high LPS concentrations, which shows the importance of lipid metabolism in the inflammatory context in general. 

Q8: Since DGAT enzymes are responsible specifically for fatty acid esterification, the authors could consider attributing this functional role other than calling it “lipid biosynthesis”. 

Q9: The authors could consider expanding the discussion on why fatty acid esterification and catabolism, apparently antagonistic pathways, have similar effects on microglia. Increased fatty acid esterification and degradation happening simultaneously seems to reflect an involvement of the glycerolipid/free fatty acid cycle, or lipid cycling(13,14). Do the transcriptomic data reveal any further aspects that could support these hypotheses? Indeed, the data on DDHD2 strengthens this conclusion as its activity is not involved in lipid cycling nor in modulating pro-inflammatory markers. 

Q10: In order to include the internal stimulus model in the discussion, the authors could perform experiments with ATGL inhibitors in APOE4/APOE4 iPSCs. If the outcomes are similar to those observed with DGAT inhibitors, the authors could suggest that lipid cycling is required to promote microglia activation in both external and internal stimuli. Alternatively, the authors could just include a discussion on lipid cycling in the discussion section in order to explore new aspects of microglial phenotype regulation.

 

References: 

1. Zadoorian A, Du X, Yang H. Lipid droplet biogenesis and functions in health and disease. Nat Rev Endocrinol. 2023 Aug;19(8):443-459. doi: 10.1038/s41574-023-00845-0. Epub 2023 May 23. PMID: 37221402; PMCID: PMC10204695. 
2. Ralhan I, Chang CL, Lippincott-Schwartz J, Ioannou MS. Lipid droplets in the nervous system. J Cell Biol. 2021 Jul 5;220(7):e202102136. doi: 10.1083/jcb.202102136. Epub 2021 Jun 21. PMID: 34152362; PMCID: PMC8222944. 
3. Huang Y, Mahley RW. Apolipoprotein E: structure and function in lipid metabolism, neurobiology, and Alzheimer’s diseases. Neurobiol Dis. 2014 Dec;72 Pt A:3-12. doi: 10.1016/j.nbd.2014.08.025. Epub 2014 Aug 27. PMID: 25173806; PMCID: PMC4253862.
4. Pires M, Rego AC. Apoe4 and Alzheimer’s Disease Pathogenesis-Mitochondrial Deregulation and Targeted Therapeutic Strategies. Int J Mol Sci. 2023 Jan 1;24(1):778. doi: 10.3390/ijms24010778. PMID: 36614219; PMCID: PMC9821307. 
5. Safieh M, Korczyn AD, Michaelson DM. ApoE4: an emerging therapeutic target for Alzheimer’s disease. BMC Med. 2019 Mar 20;17(1):64. doi: 10.1186/s12916-019-1299-4. PMID: 30890171; PMCID: PMC6425600. 
6. Sienski G, Narayan P, Bonner JM, Kory N, Boland S, Arczewska AA, Ralvenius WT, Akay L, Lockshin E, He L, Milo B, Graziosi A, Baru V, Lewis CA, Kellis M, Sabatini DM, Tsai LH, Lindquist S. APOE4 disrupts intracellular lipid homeostasis in human iPSC-derived glia. Sci Transl Med. 2021 Mar 3;13(583):eaaz4564. doi: 10.1126/scitranslmed.aaz4564. PMID: 33658354; PMCID: PMC8218593. 
7. Haney MS, Pálovics R, Munson CN, Long C, Johansson PK, Yip O, Dong W, Rawat E, West E, Schlachetzki JCM, Tsai A, Guldner IH, Lamichhane BS, Smith A, Schaum N, Calcuttawala K, Shin A, Wang YH, Wang C, Koutsodendris N, Serrano GE, Beach TG, Reiman EM, Glass CK, Abu-Remaileh M, Enejder A, Huang Y, Wyss-Coray T. APOE4/4 is linked to damaging lipid droplets in Alzheimer’s disease microglia. Nature. 2024 Apr;628(8006):154-161. doi: 10.1038/s41586-024-07185-7. Epub 2024 Mar 13. PMID: 38480892; PMCID: PMC10990924. 
8. Hickman S, Izzy S, Sen P, Morsett L, El Khoury J. Microglia in neurodegeneration. Nat Neurosci. 2018 Oct;21(10):1359-1369. doi: 10.1038/s41593-018-0242-x. Epub 2018 Sep 26. PMID: 30258234; PMCID: PMC6817969. 
9. Abud EM, Ramirez RN, Martinez ES, Healy LM, Nguyen CHH, Newman SA, Yeromin AV, Scarfone VM, Marsh SE, Fimbres C, Caraway CA, Fote GM, Madany AM, Agrawal A, Kayed R, Gylys KH, Cahalan MD, Cummings BJ, Antel JP, Mortazavi A, Carson MJ, Poon WW, Blurton-Jones M. iPSC-Derived Human Microglia-like Cells to Study Neurological Diseases. Neuron. 2017 Apr 19;94(2):278-293.e9. doi: 10.1016/j.neuron.2017.03.042. PMID: 28426964; PMCID: PMC5482419. 
10. 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. doi: 10.1038/s41593-019-0566-1. Epub 2020 Jan 20. Erratum in: Nat Neurosci. 2020 Feb;23(2):294. doi: 10.1038/s41593-020-0595-9. Erratum in: Nat Neurosci. 2020 Oct;23(10):1308. doi: 10.1038/s41593-020-0682-y. PMID: 31959936; PMCID: PMC7595134. 
11.  Benador IY, Veliova M, Mahdaviani K, Petcherski A, Wikstrom JD, Assali EA, Acín-Pérez R, Shum M, Oliveira MF, Cinti S, Sztalryd C, Barshop WD, Wohlschlegel JA, Corkey BE, Liesa M, Shirihai OS. Mitochondria Bound to Lipid Droplets Have Unique Bioenergetics, Composition, and Dynamics that Support Lipid Droplet Expansion. Cell Metab. 2018 Apr 3;27(4):869-885.e6. doi: 10.1016/j.cmet.2018.03.003. PMID: 29617645; PMCID: PMC5969538.
12. Schott MB, Weller SG, Schulze RJ, Krueger EW, Drizyte-Miller K, Casey CA, McNiven MA. Lipid droplet size directs lipolysis and lipophagy catabolism in hepatocytes. J Cell Biol. 2019 Oct 7;218(10):3320-3335. doi: 10.1083/jcb.201803153. Epub 2019 Aug 7. PMID: 31391210; PMCID: PMC6781454. 
13. Prentki M, Madiraju SR. Glycerolipid metabolism and signaling in health and disease. Endocr Rev. 2008 Oct;29(6):647-76. doi: 10.1210/er.2008-0007. Epub 2008 Jul 7. PMID: 18606873. 
14. Veliova M, Ferreira CM, Benador IY, Jones AE, Mahdaviani K, Brownstein AJ, Desousa BR, Acín-Pérez R, Petcherski A, Assali EA, Stiles L, Divakaruni AS, Prentki M, Corkey BE, Liesa M, Oliveira MF, Shirihai OS. Blocking mitochondrial pyruvate import in brown adipocytes induces energy wasting via lipid cycling. EMBO Rep. 2020 Dec 3;21(12):e49634. doi: 10.15252/embr.201949634. Epub 2020 Dec 4. PMID: 33275313; PMCID: PMC7726774.

Tags: alzheimer's disease, apoe4, lipid metabolism, microglia

doi: https://doi.org/10.1242/prelights.38200

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