ApoE4 is the strongest genetic risk factor for AD, but its mechanism of action has eluded scientists. Previous research from many labs, including from Holtzman’s, suggested that ApoE binds Aβ directly to influence its clearance (for a review, see Kim et al., 2009). These studies lacked stoichiometric measures of how much of the total peptide ApoE trapped, however. What’s more, researchers often relied on abnormally high ratios of Aβ to ApoE, said Holtzman. Now his group has quantified binding at physiological concentrations of the two proteins.

First author Philip Verghese and colleagues combined synthetic soluble Aβ, or peptide derived from human CSF or neuroglioma cells, with ApoE particles assembled with appropriate lipids, or ApoE extracted from human CSF, or mouse or human astrocytes. With these physiologically relevant sources and ApoE/Aβ ratios (around 50:1), they used ultracentrifugation and size-exclusion chromatography to measure how much Aβ bound to ApoE. They checked in the presence of H4 neuroglioma cells overproducing Aβ, too, to see if ApoE attracted the secreted peptide from cells. In all of these combinations, about 95 percent of Aβ remained free after 24 hours, regardless of ApoE isoform.

If ApoE does not directly bind Aβ in physiological fluids, then how does it modify the peptide’s clearance? The researchers looked to astrocytes, which make most of the ApoE in the central nervous system and help clear Aβ. They cultured astrocytes from ApoE knock-in and ApoE knockout mice with cell-secreted soluble Aβ. Cells without ApoE cleared more Aβ than any of the knock-ins. Of the latter, ApoE2 astrocytes cleared Aβ best, and ApoE4 cells the least. Results suggested that ApoE is unnecessary for Aβ removal by astrocytes, and actually inhibits it. The researchers confirmed this by adding ApoE particles and soluble Aβ to ApoE knockout astrocytes. Regardless of isoform, the more ApoE they added, the less the astrocytes cleared Aβ. Overall, the results imply that ApoE does not bind to Aβ, but competes for clearance somehow, for example, by binding to an Aβ receptor or receptors on the cell surface.

Could LRP1 fit that bill? Recent studies highlight the importance of LRP1 in Aβ metabolism (see Kanekiyo et al., 2011) and AD (see Harris-White and Frautschy, 2005). This receptor rests on astrocyte membranes and binds both Aβ and ApoE. There are even hints it may help clear Aβ from the vasculature (see ARF related news story). Interestingly, though Verghese and colleagues found that LRP1-deficient mouse fibroblasts removed less Aβ than usual, clearance was unaffected by ApoE. An LRP1 antibody also reduced uptake of Aβ by astrocytes. Together, these results suggest that ApoE competes for LRP1, blocking Aβ clearance.

This research says nothing about interaction with aggregated forms, said Holtzman. ApoE is found in Aβ plaques, he pointed out (see Wisniewski and Frangione, 1992). “This study looks at what might lead to Aβ aggregation in the first place,” he told Alzforum. This speaks to the difficulty in studying ApoE/Aβ interactions, since the former comes in three isoforms, different lipoprotein particles, and different lipidation states, while the latter exists as monomeric, oligomeric, and aggregated entities. “The entire problem is more complex than can be discussed in a single paper,” said Zlokovic. Previous work has suggested that ApoE is needed for clearance of aggregated Aβ, and that it facilitates Aβ clearance by astrocytes (see Koistinaho et al., 2004), he added. "With so much conflicting data, this paper suggests the field does not have a final answer," he concluded. In fact, while these new data suggest that ApoE blocks Aβ clearance, Gary Landreth and colleagues at Case Western Reserve University, Cleveland, Ohio, found that bexarotene, which enhances ApoE expression, clears Aβ and improves cognition in mice (see ARF related news story). Using brain microdialysis techniques developed in his lab, Holtzman collaborated with Landreth to show that bexarotene lifts ApoE while suppressing Aβ in interstitial brain fluid. This paper appeared earlier this month (see Ulrich et al., 2013). Bexarotene also boosts ApoE lipidation, which could explain the beneficial effects, Holtzman said.

Any explanation for how ApoE modulates Aβ clearance will have to account for the increased risk for AD that comes with ApoE4. Only further study will shed light on that question, wrote the authors. “It will be interesting to determine whether ApoE4 binds to LRP1 with higher affinity than ApoE2, functioning as a more potent antagonist of Aβ uptake,” said Steven Burden, New York University Medical School, New York. Holtzman said his group plans to investigate that.

“This is a compelling study that will definitely be controversial,” suggested Edwin Weeber, University of South Florida, Tampa. The paper thoroughly examined the ApoE/Aβ binding question, but did not address how this competition affects Aβ uptake into neurons and other cells that express LRP1, he pointed out. Nevertheless, “this study is going to change the way people think about ApoE isoforms and clearance of Aβ,” said Weeber.

Mary Jo LaDu and Leon Tai, University of Illinois at Chicago, cautioned that readers should not discount the importance of binding between ApoE and Aβ based on this paper alone. Given the unique environment of the brain, it is unclear whether in-vitro results are relevant to in vivo, LaDu said. Holtzman’s detection techniques may have missed or disrupted ApoE/Aβ complexes, she cautioned. She wondered if the researchers would find different results using an ELISA method recently developed in her lab that she believes will more likely keep complexes intact. Her group used it to detect soluble ApoE/Aβ binding in the brain tissue of humans and AD transgenic mice, as well as in human CSF (see Tai et al., 2013).

Finally, LRP1 exists in soluble form as well. It circulates in the blood, binds about 70 percent of plasma Aβ, and shepherds it to the liver for clearance, providing a natural peripheral sink. LRP1 becomes oxidized in AD patients, limiting Aβ binding and leaving more free peptide in the plasma. For that reason, Zlokovic and colleagues are working on LRP replacement as a way to reduce Aβ buildup. They previously created a recombinant fragment of one of the protein’s Aβ-binding clusters, LRPIV, and found that it curtailed Aβ pathology in Tg2576 mice and bound free peptide in human plasma (see ARF related news story). Since the protein fragment has multiple ligands, they wanted to boost its affinity for Aβ to clear the peptide more efficiently.

In their JBC paper, co-first authors Abhay Sagare, University of Southern California, Los Angeles; Robert Bell, University of Rochester Medical Center, New York; and Alaka Srivastava at ZZ Alztech, also in Rochester, made a library of LRPIV variants. They picked one, LRPIV-D3674G, that binds tightly to Aβ but weakly to other ligands such as ApoE isoforms. With five days of intravenous treatment, LRPIV-D3674G reduced endogenous brain Aβ40 and Aβ42 in Tg2576 mice about 26 percent better than the wild-type fragment. When given subcutaneously to the same mice for three months, this variant showed no toxicity and cleared Aβ40 and Aβ42 in the hippocampus, cortex, and cerebrospinal fluid by 60-80 percent. Put together with the Verghese study, this suggests that “a higher-affinity LRP fragment could promote cellular Aβ clearance and lessen ApoE-mediated slowing of Aβ removal,” said Holtzman.

With his colleagues at ZZ Alztech, Zlokovic plans to scale up the manufacturing process for this LRP fragment and submit an Investigational New Drug application with the FDA to get it on the road to clinical trials.—Gwyneth Dickey Zakaib.

References:
Verghese PB, Castellano JM, Garai K, Wang Y, Jiang H, Shah A, Bu G, Frieden C, Holtzman DM. ApoE influences Aβ clearance despite minimal ApoE/Aβ association in physiological conditions. PNAS. 2013 April 25. Abstract

Sagare AP, Bell RD, Srivastava A, Sengillo JD, Singh I, Nishida Y, Chow N, Zlokovic BV. A lipoprotein receptor cluster IV mutant preferentially binds amyloid-β and regulates its clearance from the mouse brain. J Biol Chem. 2013 Apr 11. Abstract