14 Jul 2018

As the strongest genetic risk factor for Alzheimer’s disease, ApoE, and particularly its relationship with Aβ, has been studied six ways to Sunday. More recently, scientists have turned to what the apolipoprotein does beyond Aβ. At a joint Keystone symposia—Advances in Neurodegenerative Disease Research and Therapy; and New Frontiers in Neuroinflammation—held June 17–21 in Keystone, Colorado, researchers implicated ApoE in harmful glial responses to tau pathology. In tau models, the E4 allele drove up expression of genes involved in cholesterol biosynthesis in microglia and astrocytes, while at the same time lowering expression of genes needed to export cholesterol from those cells. Other researchers reported that E4 wrought havoc in the brain even when expressed only in the periphery. In all, the meeting revealed that despite a quarter century in the research limelight, ApoE still has some secrets to give up.

In a talk, David Holtzman of Washington University in St. Louis followed up on his published data that human ApoE exacerbates tau pathology in mice (Apr 2017 conference news; Sep 2017 news). In that study, Holtzman and collaborators showed that, in the P301S tauopathy model, human ApoE intensified tau accumulation, neuronal damage, and neuroinflammation, with the E4 allele being more potent than E3 or E2. Conversely, P301S mice on an ApoE knockout background aged without neurodegeneration.

At Keystone, Holtzman attributed these effects to the secreted form of ApoE. Besides existing in different lipidation states, ApoE can also reside in different locales: Most is secreted, some is retained inside the cell. To understand the effects of extracellular ApoE, Holtzman crossed P301S mice to a strain overexpressing low-density lipoprotein receptor (LDLR), which binds to and internalizes ApoE. Holtzman had previously reported that overexpressing this receptor 10-fold in normal mice removed 90 percent of the brain’s extracellular ApoE (Dec 2009 news). The same was true when those LDLR-overexpressing animals were crossed with P301S mice, he reported. Notably, the LDLR-OE P301S animals fared similarly to P301S mice on an ApoE knockout background: They had less phospho-tau accumulation and no neurodegeneration. This suggests that it is extracellular ApoE that worsens tau pathology.

Holtzman also reported data in keeping with the idea that extracellular ApoE incites microglia. According to single-cell transcriptomics on P301S brains, clusters of cells expressing microglial activation markers expanded dramatically when the animals were between six and nine months of age. This did not happen in P301S ApoE knockouts or in P301S mice that overexpressed LDLR, suggesting again that secreted ApoE was required to rile up microglia. Overall, Holtzman interpreted his findings as more evidence that ApoE aids and abets neurodegeneration, once again casting ApoE as a therapeutic target (Apr 2018 news). 

Where did the extracellular ApoE come from? One possibility is the microglia themselves. While astrocytes are generally considered the primary source of ApoE, Holtzman reported a 100-fold spike in microglial ApoE expression in six- to nine-month-old P301S mice. In astrocytes, ApoE expression only doubled or tripled during this time period. However, Holtzman noted that astrocytes are far more abundant than microglia, and express much higher levels of the apolipoprotein in earlier disease stages than do microglia. He also cautioned that while ApoE transcripts eventually skyrocketed in microglia, to what extent that translated into ApoE protein was uncertain. In all, Holtzman still favors the idea that astrocytes are the prime source of ApoE in the brain throughout the course of disease.

There was also much chatter about ApoE’s lipidation status at Keystone. In his talk, Guojun Bu of the Mayo Clinic in Jacksonville, Florida, reported that the amount of lipids strapped to the protein varies depending on which cell type secretes it. Bu extracted astrocytes or microglia from the brains of young mice, cultured them separately, and compared the size of the ApoE particles they spat into the medium. He found that both microglia and astrocytes secreted lipidated particles, but that microglial ones were larger. Bu cautioned that the process of extracting microglia from the brain and placing them in culture is known to activate the cells, and it is unclear how closely the cultured cells resemble those in the brain (see also Part 1 of this series). Bu said he is performing a thorough lipidomics analysis of ApoE particles, comparing the lipid content of ones secreted from different cell types, and of different ApoE isoforms. In his talk, he proposed that ApoE2 particles were larger and more lipidated than ApoE4 particles, suggesting that ApoE2 might be more adept than ApoE4 at removing cholesterol from circulation, and possibly help repair axonal injuries more effectively as well.

Holtzman was skeptical that ApoE lipidation differed depending on isoform, pointing out that particles derived from human CSF look similar across genotypes. However, he was intrigued by the idea that different cell types might produce differentially lipidated particles. “The possibility that a microglia particle is different than an astrocyte particle is very high,” he said. “But it remains to be seen what those differences are, and how they matter.”

ApoE: From the Outside In?
Bu’s lab is venturing beyond the borders of the brain to investigate the effects of ApoE expression in the periphery. Unlike the AD risk factor TREM2—a receptor expressed primarily on microglia—ApoE is made throughout the body, especially in the liver. Peripheral ApoE is excluded from the blood-brain barrier, so it cannot efficiently pass into the brain. Nevertheless, Bu hypothesized that the peripheral protein might somehow influence processes in the brain.

To investigate this idea, Bu generated mice expressing either ApoE3 or ApoE4 driven by the albumin promoter to restrict expression to hepatocytes in the liver. The animals were on an ApoE knockout background and, in keeping with the apolipoprotein’s exclusion by the blood-brain barrier, Bu detected no ApoE in the brain. However, cognitive differences emerged, as albumin-E4 mice performed worse on tests of contextual fear memory than ApoE knockouts, while albumin-E3 mice performed better. When Bu crossed these animals to APP/PS1 mice, he found that compared to the ApoE knockouts, which had only diffuse plaques, albumin-E3 mice had fewer diffuse plaques, while albumin-E4 mice had more.

How could peripheral ApoE possibly influence these processes in the brain? Bu believes that peripherally expressed E4 somehow compromises the integrity of the blood-brain barrier. More dextran leaked across it in the albumin-E4 mice than in the other mice, and blood flowed more slowly through their brain arterioles, he reported at Keystone. How these vascular problems influenced myriad processes in the brain remains to be seen, though Bu speculates that neuroinflammatory responses triggered by injured vessels might play a role. The findings also exemplify the beneficial role of ApoE3, Bu told Alzforum, and argue against the idea of targeting ApoE in people who do not carry the E4 allele.

Could ApoE have crossed the barrier and seeded plaques? Bu said that so far, he has not found any ApoE in these animals’ brains. However, he did not rule out the possibility that a small amount did get in.

Some of Bu’s findings mesh with those of a previous study led by Joachim Herz of the University of Texas Southwestern Medical Center in Dallas, who reported that expression of human ApoE3 in the periphery prevented memory problems in ApoE knockout mice (Oct 2016 news). However, peripheral ApoE3 did not rescue some synaptic deficits the knockouts had. Herz also detected small amounts of human ApoE3 in the interstitial fluid, suggesting that some level of peripheral ApoE sneaks into the brain.

Bu told the audience that while he still views ApoE’s relationship with Aβ as a pivotal driver of AD risk, he thinks ApoE’s functions in multiple cell types and organs together influence the onset and course of the disease.

ApoE4: Oil Slick in a Dish?
Other researchers at Keystone presented data on ApoE’s specific effects on different cell types. In particular, several groups investigated how different ApoE isoforms swayed gene expression in iPSC-derived neurons, astrocytes, and microglia. As presented in a talk and on a poster, Alison Goate and postdoc Julia TCW of the Icahn School of Medicine at Mount Sinai, New York, generated iPSC-derived neurons, astrocytes, microglia, and microvesicular endothelial cells from 13 donors, including seven homozygous E3 carriers and six homozygous E4 carriers. They also used CRISPR gene editing to create isogenic lines from several of the donors, changing E3/E3 to E4/E4 and vice versa. At Keystone, Goate and TCW presented some of the fruits of this massive cell culture effort, reporting that compared with E3/E3 cells, E4/E4 microglia and astrocytes had dramatically elevated expression of genes involved in cholesterol biosynthesis, but reduced expression of lysosomal processing and lipid efflux genes. In other words, cholesterol synthesis was up, while the machinery needed to break it down and get rid of it was down. TCW found no elevation of cholesterol biosynthesis in iPSC-derived neurons, indicating that the pathway was only affected in glial cells.

Goate interpreted this as evidence of an uncoupling of lipid production and degradation pathways in E4 microglia and astrocytes. The findings mesh with Goate’s broader hypothesis about what drives AD, namely a defect in efferocytosis, a.k.a. the removal of dead cells and debris. Goate’s and others’ GWAS findings (see Part 1 of this series) implicate failing microglial functions such as phagocytosis in AD, and E4 appears to compound these problems by causing a lipid pileup inside cells.

John Hardy of University College London put forth a similar overarching hypothesis in his talk at Keystone. He suggested central processes that drive each neurodegenerative disease. For AD, he proposed faulty microglial clean-up of damaged membranes as a pivotal vulnerability, pointing out that many AD risk factors—such as TREM2, ABCA7, MS4A genes, and ApoE—sense and bind to lipids. Hardy added that even in the case of autosomal-dominant AD mutations in PS1, PS2, or APP, increased production of Aβ damages neuronal membranes, and ultimately microglia cannot keep up with the demand for clean-up.

In their talks at Keystone, Yadong Huang of the Gladstone Institute of Neurological Disease in San Francisco and Li-Huei Tsai of the Massachusetts Institute of Technology in Boston also described effects of ApoE isoforms on iPSC-derived human cells. Both researchers recently published their findings. In a nutshell, Huang generated iPSC-derived neurons from three E3/E3 donors and three E4/E4 donors, reporting that ApoE4 boosted both Aβ production and tau phosphorylation. Tsai, who made iPSC-derived cells and isogenic lines from a single donor, reported the same. She also found, similar to Goate and TCW, that at least in astrocytes, ApoE4 elevated expression of lipid synthesis genes, but downregulated lipid transport genes. In microglia, E4 stoked inflammatory genes, and downregulated genes involved in cell migration (see Jun 2018 news for Alzforum coverage of both papers).

While some parallels exist between Goate and Tsai’s findings, TCW commented that samples from multiple donors are needed to generate sufficient statistical power to draw conclusions. She is currently generating even more isogenic lines, in hopes they will be a useful resource for drug discovery efforts in the field.

Exemplifying an unbiased approach to exploring the brain biology of ApoE, Tal Nuriel in Karen Duff’s lab at Columbia University, New York, presented multi-omics data implicating ApoE4 in a range of cellular processes. He extracted RNA, lipids, small molecules, and proteins from aged ApoE4 or ApoE3 transgenic mouse brain, and compared isoform-dependent differences across myriad processes in AD-vulnerable regions such as entorhinal cortex, to differences in AD-resistant regions such as visual cortex. Previously, the scientists had found dysregulation of the endolysosomal pathway and an uptick in neuronal hyperactivity in AD-vulnerable brain regions in ApoE4 mice (see Nuriel et al., 2017 and Nuriel et al., 2017).

At Keystone, Nuriel added metabolic data. Differences in small-molecule metabolites in the E3 versus E4 mice hinted at alterations in mitochondrial function. To investigate this with a functional assay, Nuriel compared the oxygen consumption rate—a measure of mitochondrial function—in different brain regions of 18-20-month old E3 and E4 mice. Relative to E3 mice, E4 mice had flagging mitochondria in the cortex and hippocampus. However, the E4 entorhinal cortex, known for its vulnerability to AD, consumed more oxygen than in E3 mice. It is unclear what this means, but Nuriel suspects that neurons in the entorhinal cortex might ramp up mitochondrial function to compensate for cell stress and/or hyperactivity triggered by ApoE4. 

TCW commended the novelty and comprehensive nature of this work. Taken together, Nuriel’s, TCW’s, and other scientists’ Keystone presentations suggest that when it comes to ApoE in the brain, all cards are still on the table.——Jessica Shugart

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References

News Citations

1. ApoE and Tau: Unholy Alliance Spawns Neurodegeneration 15 Apr 2017
2. ApoE4 Makes All Things Tau Worse, From Beginning to End 20 Sep 2017
3. Mind Over Heart—LDL Receptors Crimp ApoE, Aβ Accumulation 11 Dec 2009
4. Human ApoE Antibody Nips Mouse Amyloid in the Bud 5 Apr 2018
5. A Delicate Frontier: Human Microglia Focus of Attention at Keystone 13 Jul 2018
6. Distinct Roles for Brain and Blood ApoE in Neuron Health? 6 Oct 2016
7. In Human Neurons, ApoE4 Promotes Aβ Production and Tau Phosphorylation 15 Jun 2018

Paper Citations

1. Nuriel T, Peng KY, Ashok A, Dillman AA, Figueroa HY, Apuzzo J, Ambat J, Levy E, Cookson MR, Mathews PM, Duff KE. The Endosomal-Lysosomal Pathway Is Dysregulated by APOE4 Expression in Vivo. Front Neurosci. 2017;11:702. Epub 2017 Dec 12 PubMed.
2. Nuriel T, Angulo SL, Khan U, Ashok A, Chen Q, Figueroa HY, Emrani S, Liu L, Herman M, Barrett G, Savage V, Buitrago L, Cepeda-Prado E, Fung C, Goldberg E, Gross SS, Hussaini SA, Moreno H, Small SA, Duff KE. Neuronal hyperactivity due to loss of inhibitory tone in APOE4 mice lacking Alzheimer's disease-like pathology. Nat Commun. 2017 Nov 13;8(1):1464. PubMed.

Further Reading

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