21 Oct 2023

Since the 1990s, scientists have known cholesterol is important in Alzheimer's, but they gained little traction in their efforts to understand the relationship between the lipid and the disease. Though amyloid plaques, where the sterol rubs shoulders with Aβ and ApoE, might be a place to begin, cholesterol's omnipresence has hampered progress. Every cell in the brain makes and stores it. Some even pass it around to other cells. But as seen at the 2nd Symposium on Lipids in Brain Diseases, held September 13-15 in Leiden, The Netherlands, scientists are finally getting a grip on this slippery fellow by focusing on specific metabolites and specific cells.

Anil Cashikar, from St Louis, reported that ablating 25-hydroxycholesterol (25-HC) prevents aggregation of tau in mice, while Gesine Saher from Göttingen described how mice end up with more plaques if their microglia cannot make cholesterol. From Amsterdam, next door to the historic host city, Femke Feringa reported that not only do astrocytes pass cholesterol to neurons, but that neurons hand it back—perhaps to dump toxic lipids.

Cholesterol Metabolite and Tau
Cashikar, Washington University, St. Louis, knew from previous work with Steven Paul at WashU and Wenjie Luo at Weill Cornell Medicine, New York, that cholesterol 25-hydroxylase, the enzyme that makes 25-hydroxycholesterol, ticks up in AD and in mouse models of amyloidosis and neurofibrillary tangles (Wong et al., 2020). Cashikar was curious what exactly this metabolite was doing in neurodegenerative disorders because it had been reported to promote production of interleukin-1β from microglia, and because TREM2 and ApoE, two genes upregulated in activated microglia in AD and other tauopathies, promote 25-hydroxylase activity (Wong et al., 2020). Could this enzyme, and the cholesterol derivative, contribute to microglial responses in disease?

To find out, Danira Toral-Rios in Cashikar’s lab turned to PS19 mice, which overexpress a human tau gene carrying the P301S mutation that causes frontotemporal dementia. She found that PS19 microglia express five times as much cholesterol 25-hydroxylase as do cells in wild-type mice. To see if this affects pathology, Toral-Rios mated PS19s with cholesterol 25-hydroxylase knockout (ChKO) mice. She found that 9.5-month-old crosses had much less atrophy of the piriform/entorhinal cortex than did PS19 parents. They had normal numbers of cells in the CA1 layer of the hippocampus (image at right), and of synapses in the CA3 layer. The crosses produced less phospho-tau and accumulated fewer tangles in the hippocampus and cortex, said Cashikar.

How could reducing 25-hydroxycholesterol cause such profound change? Toral-Rios found little astrogliosis and microgliosis in the crosses, suggesting inflammatory responses were dialed down. Measuring microglial markers clec7a, ApoE, and TREM2 indicated fewer disease-associated microglia in the PS19/ChKO mice than PS19 controls. Even so, the crosses had fewer homeostatic microglia than did wild-type mice, an indication that merely cutting 25-HC out of the picture does not fully restore cell homeostasis in PS19 mice.

More evidence for tempered immune responses came from transcriptomics. As per network analysis of downregulated genes, inflammatory pathways were off in the PS19/ChKO mice, including those controlled by NF-kB and Jak/Stat signaling, the latter of which is implicated in neurodegeneration in PS19 mice (Litvinchuk et al., 2018). 

How does this intersect with tau? In vitro, Cashikar’s group found that tau fibrils induce IL-1β and Cxcl10, a chemokine, in wild-type primary microglia but not microglia from the hydroxylase knockouts. IL-1β is a well-known inflammatory cytokine, while Cxcl10 is suspected of beckoning T cells into the brain (Mar 2023 news; Sep 2023 news).

Indeed, Toral-Rios found much less of the T cell CD3 in the brains of 9.5-month-old PS19/ChKO knockouts than PS19 mice. Microglia from the knockouts made more of the anti-inflammatory cytokine IL-10, whether they were challenged with tau fibrils or not. That was a surprise, said Cashikar. “It suggests that without the hydroxylase, or 25-hydroxycholesterol, microglia are generally less inflammatory,” he said.

All told, the data suggest that when microglia contact tau fibrils, they fire up inflammatory signaling in a way that involves 25-HC.

How this metabolite figures here is unclear, said Cashikar. That said, tamping it down restored normal lipid profiles. The PS19 cortex contains abnormally high levels of cerebrosides, sphingomyelins, ceramides, and phospholipids such as phosphatidylserines and phosphatidylinositols; some of these might worsen inflammation. Ceramides and sphingomyelins, for example, are known to do as much (Alessenko and Albi, 2020). Cashikar thinks blocking 25-hydroxylase activity or expression therapeutically might bring down inflammation in tauopathies.

Cholesterol and Alzheimer's
Saher’s group, at the Max-Planck Institute of Experimental Medicine, Gottingen, Germany, took a different tack. They asked what happens when they mess with cholesterol itself.

High levels of the lipid in the brain associate with amyloid plaques and risk for AD, but why that is so remains murky (Liu et al., 2020; Heverin et al., 2004; Cutler et al., 2004). 

Previous hints that cholesterol acts at the cellular level had come from meeting co-organizer Rik van der Kant's postdoc in Larry Goldstein’s lab at the University of California, San Diego. Van der Kant had found that, in neurons derived from familial AD stem cells, cholesteryl esters drove release of Aβ and accumulation of p-tau231 (Feb 2019 news). Others had reported that membrane cholesterol renders hippocampal neurons susceptible to Aβ and tau toxicity (Apr 2009 news). This implied that lowering cholesterol might slow AD, but subsequent clinical trials were negative (e.g., atorvastatin, simvastatin).

Statins may have failed because the brain synthesizes its own cholesterol. All cells in the CNS make it. Still, Saher wondered if there is a source of cholesterol that is particularly relevant to amyloidosis. To test this, Lena Spieth in the lab crossed 5xFAD mice, which develop plaques at 2 months old, with mice that had cholesterol synthesis enzymes knocked out in specific cell lineages. She examined plaques with light-sheet microscopy, in which a thin “sheet” of light excites fluorescent tags in one focal plane, reducing photobleaching and improving resolution.

Spieth found that knocking out cholesterol synthesis in 5xFAD neurons barely affected amyloid load. Amyloid precursor protein processing also proceeded apace. Might astrocytes compensate for the loss of neuronal cholesterol? Spieth looked at them next. Deleting cholesterol synthesis in astrocytes did reduce plaque load, suggesting that these glia supply neurons with the cholesterol they need to produce Aβ.

What about amyloid clearance? Scientist believe microglia phagocytose Aβ and package any they can’t degrade into dense-core plaques, compacting the overall amyloid load (May 2016 news). When Spieth shut off microglial cholesterol synthesis, amyloidosis increased and the microglia no longer rallied around plaques.

What's more, microglia failed to upregulate many of the genes they usually call upon in response to amyloid, such as Cst7, Axl, and Clec7a, suggesting that microglia need local synthesis of cholesterol to gear up. These genes are turned up in DAM, aka disease-associated microglia, known from mouse models of AD.

Scientists at the meeting liked Saher’s data. They asked her how the DAM signal was suppressed. Saher had also reported that the cholesterol precursor desmosterol tamed inflammatory microglia in models of multiple sclerosis by activating liver X receptor signaling (see Part 2 of this series), raising the question if cholesterol might do the same in AD. She thinks it is possible, but likely more complicated than in myelin disease.

Others inquired about ApoE4. “That’s a question we’ve been struggling with for 50 years,” quipped Saher. ApoE4 binds cholesterol less well than do ApoE2 or E3, but ApoE4s effects are multifacted, including changes to peripheral lipid metabolism. Two recent studies concluded that ApoE4 prevents microglia from transitioning to disease-associated states (Yin et al., 2023; Liu et al., 2023).

The Cholesterol Shuffle
Saher's data place cholesterol transport between astrocytes and neurons at the center of amyloid deposition. In her talk, Feringa, from Vrije University, Amsterdam, went a step further, reporting that transport between astrocytes and neurons is a two-way street. Not only do astrocytes pass cholesterol to neurons, as is well known, but in cell culture experiments, she found that neurons transport cholesterol to astrocytes, and also to microglia. This depends on ApoE.

Feringa used human isogenic iPSC lines engineered to carry ApoE2, 3, or 4 alleles (Schmid et al., 2021). She first compared the lipidomes of neurons, astrocytes, and microglia derived from these cells, and then asked how APOE genotype affected them. She found the three cells had substantially different lipid profiles. For example, phosphatidyl choline and ceramide predominated in neurons, cholesteryl esters and diacylglycerol in astrocytes, and lysophospholipids and ceramide derivatives in microglia. The latter had more lipids that are involved in cell signaling pathways than in metabolism or membrane structure, Feringa found. Next, ApoE genotype. APOE4 neurons contained smaller quantities of cholesteryl esters and triglycerides than did APOE3 neurons. The opposite was true of astrocytes.

Since ApoE carries lipids, Feringa thought it might help transport them in and out of these cells. To test this, she used a layer co-culture system. She grew neurons or glia in dishes, then after the cells had matured, layered cells from one culture on top of the other and assessed lipid movement a few days later. Lo and behold, when neurons were grown at the bottom with fluorescently tagged cholesterol or fatty acid, the tags ended up in astrocytes above. Neurons also transferred cholesterol to microglia. This worked only half as efficiently in ApoE4 cells.

The transport from neurons to glia surprised Feringa. “We know that once the brain has matured, astrocytes transport cholesterol to neurons via ApoE, but this data suggests the transport is less unidirectional than we thought,” Feringa told Alzforum. Because ApoE4 seems to suppress the process, she thinks it may be relevant to disease. Indeed, she ventured that the transfer may be a way to protect neurons. Some recent studies showed that if neurons accumulate reactive oxygen species, e.g., when they are hyperactive, these ROS can generate peroxidized lipids, which are toxic. “This might be a way to get rid of those lipids,” Feringa suggested.

Scientists at the meeting noted that Feringa’s data came from healthy cells, asking what might happen in a disease setting. Feringa is testing the effect of Aβ and hypoxia on the lipid transport. Others thought it important to find out how this transport happens, suggesting she look to extracellular vesicles. As for the consequences of this reverse transport, Feringa did report that when astrocytes take up cholesterol from neurons, they secrete interleukin-6. How this inflammatory cytokine affects surrounding cells remains to be seen.—Tom Fagan

Comments

No Available Comments

Make a Comment

To make a comment you must login or register.

References

Research Models Citations

1. Tau P301S (Line PS19)

News Citations

1. Neurodegeneration—It’s Not the Tangles, It’s the T Cells 17 Mar 2023
2. In 3D Cell Model of AD, Microglia and CD8+ T Cells Gang Up on Neurons 1 Sep 2023
3. Cholesteryl Esters Hobble Proteasomes, Increase p-Tau 7 Feb 2019
4. From Aging, to Aβ, to Tau—Is Cholesterol the Link in Alzheimer’s? 24 Apr 2009
5. Barrier Function: TREM2 Helps Microglia to Compact Amyloid Plaques 28 May 2016
6. Does the Brain Use Microglia to Maintain Its Myelin? 20 Oct 2023

Therapeutics Citations

1. Atorvastatin
2. Simvastatin

Mutations Citations

1. APOE C130R (ApoE4)

Paper Citations

1. Wong MY, Lewis M, Doherty JJ, Shi Y, Cashikar AG, Amelianchik A, Tymchuk S, Sullivan PM, Qian M, Covey DF, Petsko GA, Holtzman DM, Paul SM, Luo W. 25-Hydroxycholesterol amplifies microglial IL-1β production in an apoE isoform-dependent manner. J Neuroinflammation. 2020 Jun 17;17(1):192. PubMed.
2. Litvinchuk A, Wan YW, Swartzlander DB, Chen F, Cole A, Propson NE, Wang Q, Zhang B, Liu Z, Zheng H. Complement C3aR Inactivation Attenuates Tau Pathology and Reverses an Immune Network Deregulated in Tauopathy Models and Alzheimer's Disease. Neuron. 2018 Dec 19;100(6):1337-1353.e5. Epub 2018 Nov 8 PubMed.
3. Alessenko AV, Albi E. Exploring Sphingolipid Implications in Neurodegeneration. Front Neurol. 2020;11:437. Epub 2020 May 21 PubMed.
4. Liu Y, Zhong X, Shen J, Jiao L, Tong J, Zhao W, Du K, Gong S, Liu M, Wei M. Elevated serum TC and LDL-C levels in Alzheimer's disease and mild cognitive impairment: A meta-analysis study. Brain Res. 2020 Jan 15;1727:146554. Epub 2019 Nov 23 PubMed.
5. Heverin M, Bogdanovic N, Lütjohann D, Bayer T, Pikuleva I, Bretillon L, Diczfalusy U, Winblad B, Björkhem I. Changes in the levels of cerebral and extracerebral sterols in the brain of patients with Alzheimer's disease. J Lipid Res. 2004 Jan;45(1):186-93. PubMed.
6. Cutler RG, Kelly J, Storie K, Pedersen WA, Tammara A, Hatanpaa K, Troncoso JC, Mattson MP. Involvement of oxidative stress-induced abnormalities in ceramide and cholesterol metabolism in brain aging and Alzheimer's disease. Proc Natl Acad Sci U S A. 2004 Feb 17;101(7):2070-5. PubMed.
7. Yin Z, Rosenzweig N, Kleemann KL, Zhang X, Brandão W, Margeta MA, Schroeder C, Sivanathan KN, Silveira S, Gauthier C, Mallah D, Pitts KM, Durao A, Herron S, Shorey H, Cheng Y, Barry JL, Krishnan RK, Wakelin S, Rhee J, Yung A, Aronchik M, Wang C, Jain N, Bao X, Gerrits E, Brouwer N, Deik A, Tenen DG, Ikezu T, Santander NG, McKinsey GL, Baufeld C, Sheppard D, Krasemann S, Nowarski R, Eggen BJ, Clish C, Tanzi RE, Madore C, Arnold TD, Holtzman DM, Butovsky O. APOE4 impairs the microglial response in Alzheimer's disease by inducing TGFβ-mediated checkpoints. Nat Immunol. 2023 Nov;24(11):1839-1853. Epub 2023 Sep 25 PubMed.
8. Liu CC, Wang N, Chen Y, Inoue Y, Shue F, Ren Y, Wang M, Qiao W, Ikezu TC, Li Z, Zhao J, Martens Y, Doss SV, Rosenberg CL, Jeevaratnam S, Jia L, Raulin AC, Qi F, Zhu Y, Alnobani A, Knight J, Chen Y, Linares C, Kurti A, Fryer JD, Zhang B, Wu LJ, Kim BY, Bu G. Cell-autonomous effects of APOE4 in restricting microglial response in brain homeostasis and Alzheimer's disease. Nat Immunol. 2023 Nov;24(11):1854-1866. Epub 2023 Oct 19 PubMed.
9. Schmid B, Holst B, Clausen C, Bahnassawy L, Reinhardt P, Bakker MH, Díaz-Guerra E, Vicario C, Martino-Adami PV, Thoenes M, Ramirez A, Fliessbach K, Grezella C, Brüstle O, Peitz M, Ebneth A, Cabrera-Socorro A. Generation of a set of isogenic iPSC lines carrying all APOE genetic variants (Ɛ2/Ɛ3/Ɛ4) and knock-out for the study of APOE biology in health and disease. Stem Cell Res. 2021 Apr;52:102180. Epub 2021 Feb 2 PubMed.

Further Reading

No Available Further Reading