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.
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Hong Kong University of Science & Technology
This is an exciting study demonstrating that APOE4 is associated with dysregulation of cholesterol homeostasis in human but not mouse astrocytes and microglia. Although such an association was implicated in previous studies, including those addressing APOE genotype effects in peripheral cells such as macrophages, this unbiased work demonstrates that in CNS-relevant glial cells, cholesterol metabolism likely represents a major APOE4-related pathobiology in Alzheimer’s and aging brains.
It is also interesting that the defective cholesterol metabolism is unique to human cells. However, this observation should be interpreted with caution. In particular, it is known that primary cultured microglial cells behave very differently from when present in vivo. Other factors that could also impact the results, such as aging and sex, are difficult to be modeled and factored into consideration using cell culture systems.
Future studies using, for example, single cell/single nucleus RNA sequencing in animal and human brains across different APOE genotype, age and sex with or without Alzheimer-related pathologies, might offer more definitive answers in terms of relevance to humans and in-vivo environments.
View all comments by Guojun BuGladstone Institute of Neurological Disease, UCSF
This is an interesting and comprehensive whole-transcriptome study of APOE4 and Alzheimer’s disease (AD), using human iPSC-derived brain cells, mouse brain cells, and cell-type deconvoluted transcriptomic data from postmortem AD brains. An important finding is that APOE4 is associated with dysregulation of cholesterol homeostasis in human but not mouse astrocytes and microglia, suggesting a species-dependent effect of APOE4. In contrast, elevated matrisome signaling associated with chemotaxis, glial activation, and lipid biosynthesis is observed in APOE4 mixed human neuron/astrocyte culture, mouse APOE4 glial culture, and cell-type deconvoluted transcriptomic data of APOE4 glia from AD brains, suggesting that the effect of APOE4 on matrisome signaling is not species dependent.
The human-specific transcriptional effect of APOE4 stresses the importance of studying APOE4 genotype-dependent effects in human model systems. In this regard, we have shown that APOE4 is associated with increased A production in human iPSC-derived neurons, but not in mouse neurons (Wang et al., 2018), again suggesting a species-dependent effect of APOE4. With increasing evidence of species differences in various aspects of AD modeling, it seems important to test drug candidates using human iPSC-derived brain cells before moving them into clinical trials in the future.
One important question raised by this study is whether the human-specific effect of APOE4 on cholesterol homeostasis is a loss-of-function or a gain-of-detrimental-function in glial cells. It would be worth experimentally dissecting these opposite possibilities, since answering this question is crucial for potential therapeutic development based on this study.
References:
Wang C, Najm R, Xu Q, Jeong DE, Walker D, Balestra ME, Yoon SY, Yuan H, Li G, Miller ZA, Miller BL, Malloy MJ, Huang Y. Gain of toxic apolipoprotein E4 effects in human iPSC-derived neurons is ameliorated by a small-molecule structure corrector. Nat Med. 2018 May;24(5):647-657. Epub 2018 Apr 9 PubMed.
View all comments by Yadong HuangUT Southwestern Dallas
This great and very extensive study by Julia TCW et al. demonstrates that the involvement of APOE4 in AD pathology likely falls back to the key role of ApoE in lipid metabolism. Additionally, they discovered a novel gene set/pathway called “matrisome” in APOE4 and AD cases. These findings are of tremendous importance to the field, since they make us researchers think about why APOE isoforms contribute to coronary artery disease, myocardial infarction, and AD in the same isoform-specific stepwise pattern: APOE4>APOE3>APOE2. Whereas this work demonstrates potential drawbacks of using animal models for complex human diseases, it suggests that hiPSC-derived brain cell cultures provide a translatable in vitro model to study APOE4-dysregulated pathways in AD.
Please also check out the preLight article I wrote for this preprint: Cholesterol and matrisome pathways dysregulated in human APOE ε4 glia
I am excited to see it in the Sneak Peek with some additional data!
View all comments by Theresa PohlkampPicower Institute of MIT
Picower Institute for Learning and Memory
Following the posting of their preprint, Goate and colleagues have now published their work demonstrating enhanced cholesterol accumulation and decreased lipid clearance across astrocytes and microglia of APOE4 carriers. The work, which amongst its many impressive achievements examines the transcriptome of 4 cell types derived from human iPSCs across 13 individuals, uncovers a human-specific signature of dysregulated lipid metabolism that is highly variable across individual donors. How genetic heterogeneity may modify the manifestation of APOE4 phenotypes through epistatic mechanisms remains poorly understood and is of great interest to the field.
While this study exemplifies the power of employing patient-derived iPSCs to study genetic risk variants, it raises important issues regarding the variability of gene expression levels in iPS-derived cell-types across donors with broader implications for how to model GWAS variants with these technologies. Nevertheless, the work underscores the importance of cross-validation across human datasets and mouse models, and furthers our understanding of the impact of APOE4 onto lipid homeostasis and glial activation.
The Scripps Research Institute-Florida
It’s exciting to see more studies showing the importance of lipids in biological regulation. Ample evidence suggests lipids regulate membrane protein activity. Membrane proteins, on the other hand, play key roles in many important cellular signaling processes, including neuroexcitation, inflammation, amyloid processing, viral infection, and mechanosensation, etc. Hopefully, before long, the science community will realize that lipids are one of the master regulators of cellular function.
This study shows the interaction between astrocytes and microglia, and how APOE genotype affects the transportation of lipids in the brain. From the literature, cholesterol turns out to be a key factor for many aging-related diseases; to name a few, Alzheimer's disease, cardiovascular disease and diabetes. Cholesterol signaling in the brain is mainly mediated by ApoE.
This study bring us one step forward toward understanding how cholesterol metabolism is regulated in the brain and how ApoE is involved in the process. With further study of cholesterol function and regulation, we may be able to better understand the process of aging, and hopefully, find novel ways for healthy aging.
Amsterdam UMC, loc. VUmc
Maastricht University; VU University Medical Centre
Even though APOE ε4 genotype is the strongest genetic risk factor for Alzheimer’s disease, the processes through which risk is increased remain largely unclear. In this study, Julia TCW and colleagues scrutinize effects of APOE e4 on transcriptomics in human induced pluripotent cell (hiPSC) models and mice. One conclusion is that APOE knockout mice do not resemble effects associated with APOE ε4 observed human beings, suggesting that mechanistic research may perhaps best focus on human cell models.
A striking finding from the "population-based" hiPSC models from seven APOE ε3ε3 carriers with mostly normal cognition, and six APOE ε4ε4 carriers with mostly AD dementia is that the cells and astrocyte-neuron co-cultures showed mostly similar transcript patterns for ε4 versus ε3 carriers—less than 1 percent of transcripts tested differed between ε4ε4 and ε3ε3.
Importantly, the authors further demonstrate that effects of APOE ε4 on a specific cell type is person-dependent by altering APOE genotype via CRIPR-CAS for single individuals, creating so-called isogenic cell lines. The authors suggest that variability between people may reflect modification of APOE ε4 effects by other risk variants near the APOE locus (i.e., haplotypes). Their functional studies further imply that APOE ε4 may impact on lipid processing in astrocytes specifically through lysosome-dependent imbalanced cholesterol synthesis and efflux and on matrisome proteins.
The study did not investigate how amyloid and tau would influence observed transcriptomic profiles or the performed functional analyses, and so it remains to be discovered how the identified processes are related to AD disease pathophysiology. Our cerebrospinal fluid proteomics study comparing effects of APOE genotype in AD patients suggested that effects of APOE may be subtle (Konijnenberg et al., 2020), and that associated biological processes, which also included the matrisome, varied with disease stage. If individual haplotypes modify APOE ε4 effects and determine which cell types are involved, it would mean that very large sample sizes are required to tease out meaningful processes for different AD subtypes.
This study contributes to the growing evidence that patients will need therapies tailored to their causes. In order to achieve this, we need to develop more sophisticated mapping of differences between people in their underlying pathophysiological processes. APOE ε4 genotype status by itself is probably insufficient to capture such complexity.
References:
Konijnenberg E, Tijms BM, Gobom J, Dobricic V, Bos I, Vos S, Tsolaki M, Verhey F, Popp J, Martinez-Lage P, Vandenberghe R, Lleó A, Frölich L, Lovestone S, Streffer J, Bertram L, Blennow K, Teunissen CE, Veerhuis R, Smit AB, Scheltens P, Zetterberg H, Visser PJ. APOE ε4 genotype-dependent cerebrospinal fluid proteomic signatures in Alzheimer's disease. Alzheimers Res Ther. 2020 May 27;12(1):65. PubMed.
University of Pittsburgh
This exciting new study demonstrates that APOE4 is associated with dysregulation of cholesterol homeostasis in human astrocytes and microglia. The authors used various models to prove this finding, such as human iPSC-derived brain cells, mouse brain cells, and cell-type deconvoluted transcriptomic data from postmortem AD brains.
They discovered that genes that regulate cholesterol biosynthesis were upregulated in APOE4 astrocytes and microglia in contrast to the downregulation of genes associated with intracellular cholesterol trafficking (NPC1 and NPC2) and lysosomal function. Similarly, in APOE4 carriers, there was a significant decrease in the expression of genes that regulate cholesterol efflux, such as cholesterol transporters ABCA1 and ABCA7 and apolipoproteins. This decrease was accompanied by a lower level of APOE protein and cholesterol efflux from APOE4 cells that was restored by treatment with LXR agonists to APOE3 levels. The downregulation of cholesterol efflux suggests that the level of APOE-containing HDL-like lipoproteins circulating in ISF could be decreased in APOE4, potentially affecting Aβ aggregation and clearance.
The authors predicted that the increased expression of genes that regulate cholesterol biosynthesis accompanied by the downregulation of cholesterol efflux might lead to cholesterol accumulation in APOE4 astrocytes and microglia, and this was examined by filipin staining. It will be interesting to prove this finding using more accurate methods that can quantitatively assess intracellular cholesterol accumulation, such as TOF-SIMS.
A little unexpected was the result that mouse data from APOE targeted replacement mice were discordant with the data produced using human cells. One explanation may be the use of fetal tissue in mouse experiments and the difference between cholesterol metabolism in fetal and aged brains.
Another finding was the enrichment of matrisome pathway genes, i.e., extracellular matrix glycoproteins, secreted factors, and cytokines, in APOE4 cells. The authors conclude that the enrichment of the matrisome pathway increases inflammation and promotes cholesterol synthesis that, accompanied by a decreased efflux, leads to dysregulation of cholesterol homeostasis in APOE4 carriers.
University of Minnesota, Twin Cities
Despite years of intensive investigation into the role of APOE in the pathogenesis of Alzheimer’s disease, the mechanisms underlying the genetic association of the APOE4 allele with the increased risk of AD remain incompletely understood. In this comprehensive study using multiple population and isogenic lines of hiPSC-derived glial and mixed cortical models along with cell-type deconvolution of AD brain transcriptome, Julia TCW and colleagues demonstrate that APOE4 drives dysregulation of lipid metabolism in both astrocytes and microglia. While sequestration of cholesterol in lysosome escapes SREBP-mediated feedback regulation, leading to elevated cholesterol/lipid biosynthesis in APOE4 astrocytes, the crosstalk between APOE4 astrocytes and neurons causes matrisome dysregulation, triggering an inflammatory response and lipid biosynthesis, which mimics disrupted matrisome signaling in human AD brains. The study provides convincing evidence that these alterations most likely contribute to the increased AD risk associated with APOE4.
Consistent with findings from previous research, APOE4 astrocytes exhibit deficiency in APOE secretion and cholesterol/lipid efflux, and treatment with LXR agonists rescues the deficits in APOE4 astrocytes. We have shown that a unique, clinically tested HDL-mimetic peptide, 4F, enhances APOE secretion and lipidation (cholesterol/lipid efflux) in human primary astrocytes as well as mouse primary astrocytes and microglia (Chernick et al., 2018). Our preliminary studies indicate that treatment with this HDL-mimetic peptide also rescues APOE secretion and lipidation deficits in both APOE4-TR astrocytes and APOE4 hiPSC-derived astrocytes and cerebral organoids (Apr 2019 conference news; Fredriksen et al., 2021). Further experiments are underway to investigate the impact of the HDL-mimetic peptide treatment on other functional outcomes.
Intriguingly, this study reveals increased biosynthesis of terpenoid (aka isoprenoid) as well as cholesterol in APOE4 astrocytes and microglia. This is consistent with earlier findings that the levels of isoprenoids (i.e., farnesyl and geranylgeranyl pyrophosphate, FPP and GGPP), and the expression of their synthases, are elevated in AD brains (Eckert et al., 2009). The two major isoprenoids, FPP and GGPP, serve as lipid donors for an important post-translational lipid modification process of many proteins, known as protein prenylation catalyzed by prenyltransferases. The lipophilic prenyl group facilitates the anchoring of proteins in cell membranes, mediating downstream protein–protein interactions and signal transduction (Jeong et al., 2018).
Recently we have shown that protein prenylation, in particular protein farnesylation, is upregulated in human AD brains, and the genetic deletion of farnesyltransferase reduces neuropathology and rescues cognitive deficits in transgenic AD mice (Cheng et al., 2013; Jeong et al., 2021). APOE4-mediated elevation in isoprenoid biosynthesis could be another mechanism by which APOE4 increases the risk of AD.
In addition, the findings that some of the alternations in hiPSC-derived APOE4 astrocytes and microglia are not recapitulated in APOE4-TR glia have significant implications, as humanized APOE-TR/KI mice have been serving as a major approach to investigate the role of APOE in AD.
Generation of animal models that faithfully imitate human pathophysiology remains a major challenge for the research field. Whether including the human regulatory elements in the TR/KI construct improves the similarities between species awaits further investigation. While hiPSC-derived cells and organoids provide unprecedented model systems, in vivo models are indispensable as age-related systemic impacts on physiological or pathological processes can only be studied in a living organism.
References:
Cheng S, Cao D, Hottman DA, Yuan L, Bergo MO, Li L. Farnesyl Transferase Haplodeficiency Reduces Neuropathology and Rescues Cognitive Function in a Mouse Model of Alzheimer's Disease. J Biol Chem. 2013 Oct 17; PubMed.
Chernick D, Ortiz-Valle S, Jeong A, Swaminathan SK, Kandimalla KK, Rebeck GW, Li L. High-density lipoprotein mimetic peptide 4F mitigates amyloid-β-induced inhibition of apolipoprotein E secretion and lipidation in primary astrocytes and microglia. J Neurochem. 2018 Dec;147(5):647-662. Epub 2018 Nov 26 PubMed.
Eckert GP, Hooff GP, Strandjord DM, Igbavboa U, Volmer DA, Müller WE, Wood WG. Regulation of the brain isoprenoids farnesyl- and geranylgeranylpyrophosphate is altered in male Alzheimer patients. Neurobiol Dis. 2009 Aug;35(2):251-7. PubMed.
Fredriksen K, Vegoe A, O'brien T, Li L. Enhancing APOE lipidation in human iPSC-derived models with a clinically tested HDL mimetic peptide. 2021 Society for Neuroscience Annual Meeting 2021 Society for Neuroscience Annual Meeting. Virtual.
Jeong A, Suazo KF, Wood WG, Distefano MD, Li L. Isoprenoids and protein prenylation: implications in the pathogenesis and therapeutic intervention of Alzheimer's disease. Crit Rev Biochem Mol Biol. 2018 Jun;53(3):279-310. PubMed.
Jeong A, Cheng S, Zhong R, Bennett DA, Bergö MO, Li L. Protein farnesylation is upregulated in Alzheimer's human brains and neuron-specific suppression of farnesyltransferase mitigates pathogenic processes in Alzheimer's model mice. Acta Neuropathol Commun. 2021 Jul 27;9(1):129. PubMed.
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