This is Part 1 of a five-part story. See also Part 2, Part 3, Part 4, Part 5. Download a PDF of the entire series.
23 March 2012. Why has it been such a challenge to pinpoint the exact role in pathology of apolipoprotein E, which surpasses all other genetic risk factors for late onset Alzheimer’s disease? In short, because ApoE does so many things. It transports cholesterol, regulates cell signaling, promotes Aβ aggregation and/or clearance, and even acts as a neurotoxin by poisoning mitochondria. At “ApoE, Alzheimer’s and Lipoprotein Biology,” a Keystone symposium held 26 February-2 March 2012, scientists shared the latest research and brainstormed about its meaning. One take-home message: Research now seems focused on Aβ clearance, synaptic plasticity, and neurotoxicity as the three main aspects of ApoE biology. The jury is still out on which, if any, is most important. As one researcher put it, “the camps can be dogmatic and we need to integrate to keep the dialogue moving.” The cross-disciplinary nature of the meeting seemed to do that, with talks on neurophysiology, tau biology, genetics, and some of the latest clinical trials complementing presentations by the card-carrying ApoE researchers. Conference attendees uniformly found the meeting informative, and those who were unable to go can pick up highlights in this news series.
ApoE and Aβ Clearance
There is now extensive evidence, much of it from David Holtzman’s lab at Washington University, St. Louis, Missouri, that ApoE perturbs clearance of Aβ from the brain, and that ApoE4 is the most nefarious of the three isoforms in this regard. The scientists showed, for example, that Aβ has a longer half-life in PDAPP mice expressing ApoE4 than in those endowed with ApoE2 or 3 (see ARF related news story). If this is so, then reducing ApoE in the brain could prove beneficial, and, indeed, that was recently reported for all three isoforms (see ARF related news story). Do lipoprotein receptors, which bind ApoE, influence this dynamic? At Keystone, Holtzman reviewed new data on how low-density lipoprotein receptor (LDLR) plays into Aβ clearance.
Of the lipoprotein receptors regulating cholesterol, LDLR is the main one. It binds ApoE. It promotes endocytosis of apolipoproteins and their signaling (see Part 2 of this series). Does LDLR influence the half-life of ApoE? To explore this question, Holtzman and colleagues used a technique that WashU’s Randall Bateman (see ARF related news story) pioneered to measure Aβ turnover, that is, isotopic labeling with carbon-13 leucine followed by mass spectroscopy. Holtzman reported that, in mice that overexpress LDLR, production of ApoE is the same, but it turns over 2.5 times faster than normal, and the total pool of ApoE shrinks compared to control mice. Merely doubling the expression of LDLR increased Aβ clearance from the brain interstitial fluid (see ARF related news story).
Does the LDL receptor accelerate Aβ clearance because it clears ApoE, which in turn binds Aβ? To address this question, the scientists turned to cell models. Holtzman reported that the medium of cultured astrocytes that overexpress LDLR contains less ApoE, which fits with greater uptake of ApoE into these cells. In addition, the LDLR-overexpressing cells more readily subsumed Aβ added as part of conditioned medium from other cells. But as Holtzman pointed out, that alone did not clarify whether uptake of ApoE explained the cells’ appetite for Aβ, or whether the peptide might be taken up by some other pathway. Hence, his group turned to 125I-labeled Aβ to directly probe the sequence of events.
In this experiment, labeled Aβ colocalized with the LDLR in the lysosomal pathway, and LDLR stimulated not just Aβ’s uptake, but also its degradation. Intriguingly, this process seems to be independent of ApoE, Holtzman told the audience. LDLR stimulated uptake and degradation of Aβ equally in ApoE-positive and negative astrocytes. Since then, Jacob Basak in Holtzman’s lab determined that Aβ binds directly to LDLR. All told, the data from the lab suggest that LDLR acts independently of ApoE to clear Aβ, Holtzman said (see Basak et al., 2012). This is in keeping with a recent study from Spiros Georgopoulos’ group at the University of Athens, Greece. Georgopoulos found that LDLR effects on Aβ deposition in a transgenic mouse model of AD (5xFAD) are independent of ApoE (see Katsouri and Georgopoulos, 2011).
Seem straightforward so far? Don’t get used to it—no story about ApoE stays simple for long. A different interpretation of the role of ApoE4 in Aβ clearance came from Gary Landreth, Case Western Reserve University, Cleveland, Ohio. In a widely publicized study, Landreth and colleagues last month reported that bexarotene, a retinoid X receptor (RXR) agonist, boosts brain levels of ApoE in transgenic mouse models of AD while dramatically lowering soluble and insoluble Aβ and amyloid plaques (see ARF related news story and commentary). At Keystone, the data were still fresh enough to create a stir. While most in the audience seemed impressed with the findings and eagerly await the results of the first clinical trial, some niggling loose ends inevitably came into debate.
One discrepancy that raised eyebrows was that, while bexarotene seems to work wonders over the short term (two weeks), over the longer term (three months), the mice show no change in amyloid burden despite a drop in soluble Aβ in the brain. Landreth believes that plaque clearance requires a change in the activation state of microglia, and that the drug dose used might not achieve that over the long term. He told this reporter that the dose of bexarotene, which was developed as a cancer treatment, in his mouse study may be so high that it eventually desensitized glial RXRs and/or the PPARγ and liver X receptors (LXRs) with which RXRs form heterodimeric complexes. Landreth said he is planning to study the activation state of microglia after chronic treatment.
How the bexarotene data fit with ApoE as an impediment to Aβ clearance was another topic for discussion. There was consensus that the lipidation state of ApoE is crucial in this regard. Bexarotene induces a plethora of genes related to lipid metabolism, and the treated mice produce dramatic quantities of lipidated ApoE, reported Landreth. But that very multitude of RXR targets had some researchers questioning whether bexarotene works through ApoE at all. For example, at the meeting, Cheryl Wellington, from the University of British Columbia, Vancouver, Canada, presented evidence that LXR agonists reduce Aβ in ApoE4-negative mice. Landreth agreed the drug’s actions may be complex. He said he plans to tease out the relative contributions of ApoE and microglia by using ABCA1 knockouts, which fail to lipidate ApoE, and toll-like receptor 4 mutants, which do not activate microglia.
C2N, a diagnostics company founded by Holtzman and Bateman, is collaborating with Case Western on a randomized, placebo-controlled clinical trial for bexarotene that will begin enrolling volunteers in the second quarter of 2012. Landreth told this reporter that it is a small pilot study to look at CSF changes in Aβ and ApoE. Participants must be ApoE4 negative. “If we see an effect in CSF, then we will consider pursuing this further,” he told ARF. A notice on his lab’s home page says that no new patients can be enrolled for this small trial.
In her presentation, Wellington emphasized the role of ApoE lipidation. Wellington reminded the audience that ABCA1 (ATP-binding cassette A1), a cell-surface cholesterol and phospholipid transporter that transfers lipids onto ApoE, plays a key role in formation of lipoprotein particles. She noted that ABCA1 knockouts retain much less ApoE in the brain, and deposit more Aβ than controls (see ARF related news story), whereas overexpressing ABCA1 in PDAPP mice protects them against Aβ accumulation. Poorly lipidated ApoE slows Aβ clearance, while lipidating the protein “greases the wheels,” she concluded. Wellington’s take was that liver X receptor agonists, which, like bexarotene, induce ABCA1 and therefore promote lipidation of ApoE, may help clear Aβ from the brain. Lipidation, then, could resolve some of the contradictions on whether lowering or elevating brain ApoE would be therapeutic.
This story being about ApoE, it’s now time to add another layer of complexity. What about the role of ApoA1, the major apolipoprotein in the body’s periphery. ApoA1 is the major protein in high-density lipoproteins (HDL) that carry the “good cholesterol” in the bloodstream. HDLs protect against cardiovascular disease, and there is considerable interest in understanding whether circulating HDL may also protect against AD, said Wellington. Researchers previously reported that ApoA1 inures the vasculature against cerebral amyloid angiopathy (CAA), aka deposition of Aβ in the brain’s blood vessels. Overexpressing ApoA1 in transgenic AD mouse models prevents this vascular pathology, while ApoA1 knockouts are more susceptible to it (see ARF related news story). Like ApoE, ApoA1 receives lipids from ABCA1, suggesting that LXR agonists might boost vascular clearance of Aβ through lipidating ApoA1 and boosting HDLs.
While that may turn out to be true, there is a twist. Wellington reported that the commonly used LXR agonist GW3965 increases ApoA1 levels in brain and plasma of APP/PS1 mice; however, in ABCA1-negative animals, it only boosts brain ApoA1, not plasma. The finding suggests that distinct mechanisms regulate ApoA1 in the brain and in the periphery, said Wellington. It is unclear how LXR agonists boost brain ApoA1, but the mechanism might offer new insight into Aβ clearance and explain the link between cardiovascular health and AD, she said.
If a predominantly peripheral apolipoprotein may move Aβ in the brain, what about the role of ApoE in the plasma? In a short talk, Huntington Potter, University of South Florida, Tampa, showed how he addressed this using parabiosis. In this technique, two different animals are surgically connected so that they share a circulatory system. Potter revealed that when APP/PS1 ApoE+/- mice are conjoined with APP/PS1 ApoE nulls, the number of Aβ plaques in the latter, even though low to begin with, drop significantly. The parabiosis corrected hypercholesterolemia in the transgenic mice as well. Potter said that none of the ApoE in circulation gets into the mouse brain. He concluded that increasing plasma ApoE could be a potential strategy for reducing Aβ in the brain.
Continuing the vascular theme, Guojun Bu at the Mayo Clinic, Jacksonville, Florida, outlined a potential role for blood vessels’ smooth muscle cells in Aβ metabolism. Bu studies the role of low-density lipoprotein-related protein 1 (LRP1), a member of the LDLR family. His lab previously reported that conditionally knocking out LRP1 in the mouse forebrain neurons sparks a plethora of bad events. Synaptic markers go, as do dendritic spines; neuroinflammation flares up, and motor control and cognition decline. The working hypothesis is that these deficits are due to altered lipid metabolism (see ARF related news story). Strong evidence also implicates the lipoprotein receptor in Aβ clearance (for a review see Zlokovic et al., 2010). This could be relevant to AD, suggested Bu, because people with the disease appear to have too little LRP1 in blood vessels (see Bell and Zlokovic, 2009). If cells of the vasculature take up Aβ via LRP1, then loss of the receptor could impede Aβ’s removal from the brain through the vessels. However, which cells of the blood vessels might employ that mechanism is unclear.
To get a handle on this, Bu and colleagues conditionally knocked out LRP1 only in smooth muscle cells of the vasculature. In wild-type knockouts, endogenous Aβ40 and Aβ42 rose in the brain. In APP/PS1 transgenic mice, total Aβ40 and 42 rose, as did the plaque burden. The findings suggest that smooth muscle cells in the vasculature contribute to clearance of Aβ, Bu told the audience. The finding set the audience abuzz. Some researchers said it helps clarify how Aβ is transported out of the brain. On that, scientists have debated whether endothelial cells are prime movers of the peptide. But, in fact, Bu hinted at new evidence that endothelial LRP has little impact on Aβ clearance from the brain. In any event, the mouse genetic data indicate that LRP1 in brain vasculature smooth muscle cells plays an important role in Aβ clearance. Bu said that Aβ may downregulate LRP1 in smooth muscle cells, potentially setting off a vicious cycle that blocks Aβ clearance. Bu, Holtzman, and meeting co-organizer Joachim Herz co-wrote a chapter on ApoE for The Biology of Alzheimer Disease on the current state of research in the field (see ARF book review).—Tom Fagan.
This is Part 1 of a five-part story. See also Part 2, Part 3, Part 4, Part 5. Download a PDF of the entire series.