The issue of whether to increase or decrease ApoE levels for AD therapy is confounded by the multiple functions of ApoE. In a landmark paper, Landreth and coworkers clearly demonstrate that bexarotene treatment, an RXR agonist, increases endogenous mouse ApoE levels, enhancing soluble Aβ clearance within hours, in APP/Aβ transgenic mice. Further, in this significant paper, bexarotene rapidly reduced Aβ plaque burden by greater than 50 percent in 72 hours and reversed cognitive deficits, and thus represents a novel AD therapy. These data are in apparent contrast with studies in which Aβ deposition is greatest with mouse-ApoE compared to the lack of ApoE (ApoE-/-) in multiple APP/Aβ transgenic models (e.g., publications from Fagan/Holtzman and Bales/Paul). However, these same studies further demonstrate that amyloid burden from greatest to least is: mouse-ApoE, ApoE-/-, ApoE4. Thus, one perspective on this issue of whether a therapeutic approach should increase or decrease ApoE is to compare ApoE4 to ApoE-/-. While ApoE4 has a beneficial effect on Aβ deposition compared to ApoE-/-,...  Read more

The issue of whether to increase or decrease ApoE levels for AD therapy is confounded by the multiple functions of ApoE. In a landmark paper, Landreth and coworkers clearly demonstrate that bexarotene treatment, an RXR agonist, increases endogenous mouse ApoE levels, enhancing soluble Aβ clearance within hours, in APP/Aβ transgenic mice. Further, in this significant paper, bexarotene rapidly reduced Aβ plaque burden by greater than 50 percent in 72 hours and reversed cognitive deficits, and thus represents a novel AD therapy. These data are in apparent contrast with studies in which Aβ deposition is greatest with mouse-ApoE compared to the lack of ApoE (ApoE-/-) in multiple APP/Aβ transgenic models (e.g., publications from Fagan/Holtzman and Bales/Paul). However, these same studies further demonstrate that amyloid burden from greatest to least is: mouse-ApoE, ApoE-/-, ApoE4. Thus, one perspective on this issue of whether a therapeutic approach should increase or decrease ApoE is to compare ApoE4 to ApoE-/-. While ApoE4 has a beneficial effect on Aβ deposition compared to ApoE-/-, ApoE4 has a negative effect on LTP and Aβ-induced neurotoxicity. Indeed, the magnitude of LTP in both the absence (1) and presence (2) of oligomeric Aβ is greater with ApoE-/- mice compared to ApoE4 targeted replacement mice ex vivo. Furthermore, neuronal survival after oligomeric Aβ treatment is greatest in neurons co-cultured with glia isolated from ApoE-/- mice compared to neurons with glia from ApoE4 targeted replacement mice (3). Thus, although raising ApoE levels may exert a beneficial function on Aβ deposition, ApoE4 may also represent a toxic gain of function. Therapeutics that target specific aspects of ApoE function, of which bexarotene should be considered, may prove to be the most beneficial approach for AD.

References:
1. Trommer BL, Shah C, Yun SH, Gamkrelidze G, Pasternak ES, Ye GL, Sotak M, Sullivan PM, Pasternak JF, LaDu MJ: ApoE isoform affects LTP in human targeted replacement mice. Neuroreport 2004, 15:2655-2658. Abstract

2. Trommer BL, Shah C, Yun SH, Gamkrelidze G, Pasternak ES, Stine WB, Manelli A, Sullivan P, Pasternak JF, LaDu MJ: ApoE isoform-specific effects on LTP: blockade by oligomeric amyloid-beta1-42. Neurobiology of disease 2005, 18:75-82. Abstract

3. Manelli AM, Bulfinch LC, Sullivan PM, LaDu MJ: Abeta42 neurotoxicity in primary co-cultures: effect of ApoE isoform and Abeta conformation. Neurobiol Aging 2007, 28:1139-1147. Abstract

View all comments by Mary Jo LaDu
View all comments by Leon Tai

The data presented in this paper show the most rapid removal of Aβ of any systemically applied therapy. They hold great promise for testing the amyloid hypothesis, and possibly for application in secondary prevention trials of asymptomatic patients where anti-amyloid therapies are most likely to be effective. However, reasonable dosages need to be identified before large-scale human applications can be considered. The extreme doses used in cancer studies did indicate some severe reactions to the agent, albeit rare. Our center (Byrd Alzheimer Institute) will not be writing prescriptions at this time, but look forward to well-controlled clinical trial opportunities.

View all comments by Dave Morgan

Some LXR ligands interact directly with γ-secretase. The structure of bexarotene suggests that it, too, may have a direct interaction with the complex. Given the rapidity of the effects observed, it would be interesting to see data for this compound in a cell-free γ-secretase assay.

Whilst bexarotene is indeed FDA approved for oncology, the safety profile is unlikely to translate to chronic administration in an elderly population—particularly not at equivalent doses to those used. This would indicate that more potent/bio-available analogues will be required. Understanding the exact properties of the drug with regard to amyloid reduction will be critical.

References:
Czech C, Burns MP, Vardanian L, Augustin A, Jacobsen H, Baumann K, Rebeck GW. Cholesterol independent effect of LXR agonist TO-901317 on γ-secretase. J Neurochem. 2007 May;101(4):929-36. Abstract

View all comments by Adam Kline

I think this is a very intriguing paper. Most therapeutic programs have targeted amyloid peptide production, or immune-mediated clearance of Aβ. This novel strategy targets ApoE-mediated clearance of Aβ through activation of the retinoid X receptors. Since ApoE is by far the strongest genetic risk factor in sporadic AD, it is reasonable to assume that ApoE-related therapeutics may be disease modifying. That idea is backed by strong experimental data from studies in animal models. And since bexarotene is already FDA approved for another indication, it may advance rapidly into human studies.

So this is very exciting work indeed. However, mouse models have not reliably predicted therapeutic effects in humans. Caution is crucial until human studies confirm target engagement (enhanced amyloid peptide clearance). Trials have already shown that two monoclonal antibodies reduce brain amyloid load as seen by PET imaging, so those agents may be more likely to succeed. However, the activity of bexarotene should be straightforward to confirm by looking for changes in the cerebrospinal...  Read more

I think this is a very intriguing paper. Most therapeutic programs have targeted amyloid peptide production, or immune-mediated clearance of Aβ. This novel strategy targets ApoE-mediated clearance of Aβ through activation of the retinoid X receptors. Since ApoE is by far the strongest genetic risk factor in sporadic AD, it is reasonable to assume that ApoE-related therapeutics may be disease modifying. That idea is backed by strong experimental data from studies in animal models. And since bexarotene is already FDA approved for another indication, it may advance rapidly into human studies.

So this is very exciting work indeed. However, mouse models have not reliably predicted therapeutic effects in humans. Caution is crucial until human studies confirm target engagement (enhanced amyloid peptide clearance). Trials have already shown that two monoclonal antibodies reduce brain amyloid load as seen by PET imaging, so those agents may be more likely to succeed. However, the activity of bexarotene should be straightforward to confirm by looking for changes in the cerebrospinal fluid ApoE and Aβ. If the mouse pharmacodynamic effect is confirmed, bexarotene could be an excellent candidate for studies in prodromal and preclinical AD.

View all comments by Paul Aisen

This is very exciting work. With the RXR agonist bexarotene, a drug approved by the FDA, three different AD mouse models clear amyloid-β and regain cognitive function. It is amazing that it works so fast and dramatically. However, none of these models has tau pathology. How does it affect tau pathology? Before, we also had some good candidates that reduced Aβ plaque and rescued cognitive impairment in animal models, but in clinical trials, they failed. So we look forward to the next clinical trial with bexarotene.

Also, ApoE4 increases the risk of developing the late-onset form of AD, and a lot of studies have shown that ApoE4 is toxic and that more ApoE4 leads to more plaques. If bexarotene enhances the expression of ApoE, then AD patients with E4 alleles will have more toxic ApoE4. I wonder if bexarotene will be beneficial to those patients. It has also been reported that ApoE protein level decreases on bexarotene. If so, then bexarotene may be of benefit. The effect of bexarotene probably depends on which is more important, the decrease of ApoE or the toxicity of ApoE4.

View all comments by Jianhua Shi

Before we break out the champagne, let us be aware that mouse ApoE and human ApoE are not the same!

References:
Maloney B, Ge YW, Alley GM, Lahiri DK. Important differences between human and mouse APOE gene promoters: limitation of mouse APOE model in studying Alzheimer's disease. J Neurochem. 2007 Nov;103(3):1237-57. Abstract

View all comments by John Wojdyla

The report by Cramer et al. is potentially exciting because it offers a plausible explanation as to why ApoE4 should be a risk factor for AD, and more importantly suggests that bexarotene, an off-the-shelf retinoid X receptor agonist, may prove to be an effective treatment for the disease. Unfortunately, our results with bexarotene treatment in the APPSwFILon, PSEN1*M146L*L286V (5XFAD) mouse model of Alzheimer's disease failed to provide positive results. This 5XFAD model is one of the most aggressive of all AD models, with Aβ deposits appearing as early as eight weeks after birth. It may be that the high rate of Aβ deposition in this model overwhelms the ability of bexarotene to stimulate clearance. However, it does indicate that testing should be done in other AD transgenic models to gain a fuller picture of what is taking place.

We started to feed the 5XFAD mice at seven to eight weeks of age with chow containing 500 mg/kg bexarotene, which corresponds to a consumption of 2 mg per day or an oral dose of 100 mg/kg/day (n = 8). Control 5XFAD mice were fed normal chow (n =...  Read more

The report by Cramer et al. is potentially exciting because it offers a plausible explanation as to why ApoE4 should be a risk factor for AD, and more importantly suggests that bexarotene, an off-the-shelf retinoid X receptor agonist, may prove to be an effective treatment for the disease. Unfortunately, our results with bexarotene treatment in the APPSwFILon, PSEN1*M146L*L286V (5XFAD) mouse model of Alzheimer's disease failed to provide positive results. This 5XFAD model is one of the most aggressive of all AD models, with Aβ deposits appearing as early as eight weeks after birth. It may be that the high rate of Aβ deposition in this model overwhelms the ability of bexarotene to stimulate clearance. However, it does indicate that testing should be done in other AD transgenic models to gain a fuller picture of what is taking place.

We started to feed the 5XFAD mice at seven to eight weeks of age with chow containing 500 mg/kg bexarotene, which corresponds to a consumption of 2 mg per day or an oral dose of 100 mg/kg/day (n = 8). Control 5XFAD mice were fed normal chow (n = 7). Mice were sacrificed at 21, 31, and 50 days after feeding commenced. We found no difference between test and control mice in a standard Morris water maze test, and no significant difference in the number of Aβ deposits.

Transgenic mouse models of Alzheimer's disease vary widely in their properties. The model we studied may be an unsatisfactory representation of AD pathology. However, the results do illustrate that the phenomenon described by Cramer et al. is not entirely straightforward. More in-depth investigation is clearly indicated.

View all comments by Jian-Ping Guo
View all comments by P.L. McGeer
View all comments by Claudia Schwab

Reply to comment by Jian-Ping Guo, P.L. McGeer, and Claudia Schwab
We communicated the results of our study in advance of publication with these authors.

It is important that oral administration of bexarotene employ the micronized form of the drug (Targretin™, Eisai, Inc.) due to the efficiency of absorption in the gut. The FDA filing documents a sixfold difference in efficacy compared to the powdered form of bexarotene.

Dr. Guo et al. note that their model might be too robust to observe an effect of bexarotene. We tested the effect of bexarotene in a similarly aggressive model of AD, the APP/PS1-21 mice. A 20-day treatment resulted in a reduction of soluble Aβ and an approximately 35 percent decrease in plaque number. Importantly, we found improved behavior in both the Morris water maze and contextual fear conditioning. Thus, I don't think that this consideration explains their results.

The correspondents noted that they did not find a change in plaque load in drug-treated mice. Indeed, we observed this, too. One of the surprising findings was that...  Read more

Reply to comment by Jian-Ping Guo, P.L. McGeer, and Claudia Schwab
We communicated the results of our study in advance of publication with these authors.

It is important that oral administration of bexarotene employ the micronized form of the drug (Targretin™, Eisai, Inc.) due to the efficiency of absorption in the gut. The FDA filing documents a sixfold difference in efficacy compared to the powdered form of bexarotene.

Dr. Guo et al. note that their model might be too robust to observe an effect of bexarotene. We tested the effect of bexarotene in a similarly aggressive model of AD, the APP/PS1-21 mice. A 20-day treatment resulted in a reduction of soluble Aβ and an approximately 35 percent decrease in plaque number. Importantly, we found improved behavior in both the Morris water maze and contextual fear conditioning. Thus, I don't think that this consideration explains their results.

The correspondents noted that they did not find a change in plaque load in drug-treated mice. Indeed, we observed this, too. One of the surprising findings was that chronic drug treatment of APP/PS1 mice was associated with an immediate fall in plaque burden, which then returned to the levels observed in vehicle-treated mice. We found improved behavior in the same two behavioral assays after 90 days of treatment. One possible conclusion from this experiment is that plaques don't matter, and we are actively pursuing this issue.

It should also be noted that in the periphery, there is robust induction of P450 enzymes with sustained bexarotene administration. It is possible that the dose of drug required to maintain microglia in an "alternatively activated" (M2) state, which is anti-inflammatory and conducive to plaque phagocytosis, is greater than that necessary to drive ApoE/HDL induction. The sustained drug treatment may result in efficient drug degradation, lowering the CNS and plasma levels of bexarotene. We are now testing this possibility.

We couldn't agree more that further in-depth study is necessary, and are anxious to have our studies repeated in others' hands.

View all comments by Gary Landreth

Carriers of the detrimental ApoE4 genotype have long been hypothesized to have slower Aβ catabolism, leading to earlier onset and more amyloid burden than non-ApoE4 carriers. Critical questions involve defining the mechanisms by which ApoE4 leads to sluggish Aβ turnover so that therapeutic methods to correct this can be developed. Brad Hyman and colleagues add to the evidence that ApoE is a critical modulator of Aβ metabolism.

Using AD brains of various ApoE genotypes that were carefully matched for amyloid burden, this group characterized the soluble fraction and observed significantly more higher-molecular-weight Aβ by direct visualization on SDS gels and after separation by size exclusion chromatography (SEC). Therefore, soluble Aβ is independent of amyloid burden, and the proportion of soluble oligomeric Aβ species is higher in ApoE4 carriers. Remarkably, ApoE co-eluted with Aβ during SEC and immunoprecipitated with Aβ, and it is possible that the ApoE-Aβ association makes specific Aβ epitopes under native conditions.

In a key advance, Hyman and colleagues...  Read more

Carriers of the detrimental ApoE4 genotype have long been hypothesized to have slower Aβ catabolism, leading to earlier onset and more amyloid burden than non-ApoE4 carriers. Critical questions involve defining the mechanisms by which ApoE4 leads to sluggish Aβ turnover so that therapeutic methods to correct this can be developed. Brad Hyman and colleagues add to the evidence that ApoE is a critical modulator of Aβ metabolism.

Using AD brains of various ApoE genotypes that were carefully matched for amyloid burden, this group characterized the soluble fraction and observed significantly more higher-molecular-weight Aβ by direct visualization on SDS gels and after separation by size exclusion chromatography (SEC). Therefore, soluble Aβ is independent of amyloid burden, and the proportion of soluble oligomeric Aβ species is higher in ApoE4 carriers. Remarkably, ApoE co-eluted with Aβ during SEC and immunoprecipitated with Aβ, and it is possible that the ApoE-Aβ association makes specific Aβ epitopes under native conditions.

In a key advance, Hyman and colleagues demonstrate that physiologically lipidated ApoE4 increases Aβ oligomerization in vitro. This appears to be a specific structural property of extracellular ApoE4. They first purified secreted ApoE particles from immortalized murine astrocytes expressing human ApoE2, ApoE3, or ApoE4, mixed these with synthetic Aβ, and observed that oligomer levels were significantly greater in the presence of ApoE4, which seems to stabilize the oligomers compared with ApoE2 and ApoE3. That Aβ oligomerization follows an ApoE4->ApoE3->ApoE2 relationship was confirmed using a split-luciferase assay in which Aβ oligomerization is required to observe luminescence. Furthermore, Aβ purified from the soluble fraction of AD patient brains forms high-molecular-weight Aβ species when incubated with ApoE. Importantly, ApoA-II, which is another apolipoprotein found on brain HDL particles, did not increase Aβ oligomerization using the split-luciferase assay. No effects on Aβ oligomerization were observed in cell lysates, suggesting that ApoE specifically affects Aβ in the extracellular milieu. Finally, reducing a salt bridge in ApoE4 by converting the arginine at amino acid 61 to a threonine reduced the level of Aβ oligomerization back to the level of ApoE3. This finding provides further support that the structural differences caused by arginine 61 specifically in human ApoE4 may be at the root of ApoE4’s dysfunction.

Further experiments explored the selectivity of the effect on Aβ oligomerization by other lipoproteins. Both ApoA-I and ApoJ led to significant increases in Aβ oligomers in the split-luciferase assay, albeit far more modestly than ApoE4. Hyman’s group then demonstrated that the lipid binding C-terminal domain of ApoE is necessary and sufficient for Aβ oligomerization.

Finally, in a series of elegant experiments, the Hyman group demonstrated that human ApoE purified from brain tissue affects Aβ oligomerization in an isoform-dependent manner, with ApoE4->ApoE3. Immunodepletion of ApoE from these samples significantly reduced Aβ oligomerization.

What is critically important about these experiments is that the researchers used sources of ApoE that are physiologically lipidated, unlike innumerable previous studies that used either unlipidated ApoE or ApoE reconstituted with non-physiological lipids. However, the ApoE-containing HDL particles used in these experiments can be considered “baseline” with respect to their lipid content. This is an important point, as modifying the lipidation status of ApoE has been under considerable study as a therapeutic approach for AD. For example, agonists of Liver-X-Receptor (LXR) and Retinoid-X-receptor (RXR) nuclear receptors, increase the expression of genes such as ABCA1 and ABCG1 that physiologically add lipids to apolipoprotein acceptors including ApoE. These agonists consistently improve cognitive behavior in APP-expressing mice. The studies vary in terms of the efficacy of these agonists to reduce amyloid or Aβ levels, or to shift Aβ from the insoluble to the soluble fraction. Furthermore, ABCA1, which is the rate-limiting step in apolipoprotein lipidation, is a key player in Aβ metabolism in vivo, as deletion of ABCA1 slows Aβ turnover, whereas selective overexpression of ABCA1 in the brain nearly eliminates amyloid deposits. What is not yet known is the relationship between ApoE lipidation and ApoE structure, particularly with respect to the argining 61 salt bridge. Whether LXR/RXR agonists or genetic manipulation of ABCA1 activity affect ApoE structure, thereby affecting Aβ oligomer formation, stability, or degradation, is not fully understood.

An important caveat to the studies using LXR agonists and genetically modified ABCA1 murine lines is that they have thus far been conducted on murine ApoE. Whether these pathways rescue the deficits in human ApoE4 with respect to Aβ metabolism, or whether they enhance the detrimental effects of ApoE4, is now a crucial question to address.

View all comments by Cheryl Wellington