Sadowski and colleagues previously showed that an Aβ fragment (residues 12-28) containing solely the ApoE binding domain can complex with ApoE in vitro and prevent it from driving formation of toxic Aβ fibrils (see Sadowski et al., 2004). Now they have adapted this peptide for in vivo application, switching valine 18 for proline to make it non-fibrillogenic, using all D-stereoisomers to make it less biologically active, and protecting the N- and C-terminals by acetylation and amidation, respectively. The modifications generated a peptide, designated Aβ12-28P that has decreased immunogenicity and extended serum half-life.

Before testing the peptide in AD mouse models, Sadowski and colleagues made sure that it could still bind to ApoE. They found that though it does not bind as strongly as the native Aβ12-28 in vitro, the dissociation constant for the ApoE interaction is still very low (Kd is 32 +/- 5 nM), giving an inhibition constant of 13 nM, which is in the therapeutic range. Next, the researchers tried the inhibitor in single (APPSwe) and double (APPSwe/PS1) transgenic mice. They administered the drug for six months, three times a week by intraperitoneal injection, starting when Aβ deposits first appear in these models (age 12 months for single, and two months for double transgenics). After the treatment period the researchers found that total Aβ deposits, as determined from immunohistochemical analysis with two different antibodies (6E10 and 4G8), were reduced by about 50 and 45 percent in the neocortex and hippocampus, respectively, of both transgenic strains. Thioflavin S staining revealed that fibrillar Aβ was also reduced by 38 and 32 percent, respectively in the neocortex and hippocampus of single transgenics, and 22 and 32 percent in double transgenics. They found that ApoE deposition within Aβ plaques was also significantly reduced, again by about 50 percent in the two different mouse strains.

As the authors point out, one potential drawback to therapies that prevent aggregation of Aβ is that they might increase levels of soluble oligomeric forms of the peptide, which are now generally considered to be the most toxic (see ARF related news story). But the researchers found that levels of Aβ in formic acid extracts, representing soluble forms of the peptide, were also reduced. Soluble Aβ40 and 42 were lower by 25 percent in APPSwe mice and 44 and 32 percent, respectively, in APPSwe/PS1 transgenic animals. The authors also found that the reduction in Aβ load was accompanied by behavioral improvements. In a radial arm maze test of working memory, treated animals performed just as well as wild type, while untreated transgenic animals had significant reduction in working memory capability, making significantly greater numbers of errors.

The results indicate that Aβ12-28P does indeed block ApoE driven aggregation and deposition of Aβ in vivo and that this protects the animals against memory loss. But there may be other possible modes of action, including induction of an immune response or alteration of cholesterol levels (ApoE is a major cholesterol binding protein in the brain). That the researchers found a transient increase in total serum cholesterol following a single i.v. injection of Aβ12-28P suggests that alteration of lipid profiles might be linked to the effects of the treatment. However, Aβ antibody profiles were no different between treated and untreated animals, indicating that the peptide does not spur a strong immune reaction. Though active and passive immunotherapies are, of course, being actively pursued as potential therapies in their own right (see ARF related news story), there are some concerns that this strategy might clear parenchymal plaque burden but contribute to increased deposits of Aβ in the blood vessel deposits, potentially leading to increased cerebral amyloid angiopathy (see ARF related news story). But Sadowski and colleagues found that in APPSwe mice, Aβ12-28P reduced Aβ deposits in cortical blood vessels by 70 percent. “This observation demonstrates an additional therapeutic benefit of blocking the ApoE/Aβ interaction that has not been observed with immunization against Aβ,” write the authors.

As for intraneuronal Aβ, which may be the most toxic form to neurons (see ARF related news story), the report by Bu and colleagues in the November 24 Journal of Biological Chemistry, shows that ApoE and the low density lipoprotein receptor-related protein (LRP) have a hand in boosting that particular cache of Aβ.

The LRP receptor is expressed in the soma and dendrites of neurons and is involved in internalizing many different ligands, including ApoE. It also binds to Aβ. The role of LRP in AD is still somewhat debatable, with some studies suggesting that the receptor aids in Aβ clearance(see ARF related news story) and other suggesting that it promotes Aβ accumulation. On the latter score, Bu and colleagues have shown that overexpression of a mini human LRP receptor (mLRP2), which behaves by all counts like the full length, in the PDAPP mouse model of AD leads to increased levels of soluble Aβ and compromised memory performance (see ARF related news story). That study was done in aged, 22 month old mice. Now Bu and colleagues address what happens in young mice before plaque deposits are apparent, a fundamental question in the study of AD etiology and pathology.

First author Celina Zerbinatti and colleagues report that membrane-bound (Triton X-100 extractable) and insoluble (guanidine solubilizable) Aβ42 were significantly elevated (by 20 and 12 percent, respectively) in PDAPP/mLRP2 mice compared to PDAPP controls. “Although the effects are small, they demonstrate the involvement of LRP in regulating brain Aβ levels,” write the authors. Because they found no changes in APP levels or APP processing (CTF-β levels were unchanged) it is tempting to conclude that the increased Aβ levels resulted from reduced clearance. In support of this idea the authors found that levels of CSF Aβ were significantly decreased in PDAPP/mLRP2 vs PDAPP mice.

To determine exactly where Aβ was ending up, the authors used confocal microscopy to colocalize the peptide with cell markers. They found that in PDAPP animals Aβ42 localized with the neuronal marker NeuN and also with the lysosomal marker LAMP-1, suggesting that the peptide in located internally with lysosomes. Interestingly, the researchers also found significant loss of NeuN-colocalized Aβ42 in mice lacking ApoE compared to animals homozygous for the lipoprotein gene, suggesting that ApoE may contribute to the accumulation of intraneuronal Aβ. “Our results support the hypothesis that LRP binds and endocytosis Aβ42 both directly and via ApoE but that endocytosed Aβ42 is not completely degraded and accumulates in intraneuronal lysosomes,” write the authors.

The demonstration that PC12 cells clear Aβ from cell culture medium more rapidly if the cells also expressed mLRP2 adds weight to that argument. That ApoE3 or ApoE4 particles also increased that clearance suggests that blocking the ApoE/Aβ interaction, as with Sadowski’s Aβ peptide, may help reduce intraneuronal Aβ, too.—Tom Fagan.

References:
Sadowski MJ, Pankiewicz J, Scholtzova H, Mehta PD, Prelli F, Quartermain D, Wisniewski T. Blocking the apolipoprotein E/amyloid-beta interaction as a potential therapeutic approach for Alzheimer’s disease. PNAS. Dec 5, 2006;103:18787-18792. Abstract

Zerbinatti CV, Wahrle SE, Kim H, Cam JA, Bales K, Paul SM, Holtzman DM, Bu G. Apolipoprotein E and low density lipoprotein receptor-related protein facilitate intraneuronal Abeta42 accumulation in amyloid model mice. J. Biol. Chem. November 24, 2006;281:36180-36186. Abstract