12 Feb 2016

Bexarotene has shaped up to be a jack-of-all-trades in Alzheimer’s—various reports suggest that it clears plaque by boosting ApoE expression or microglia, others that it stimulates neurogenesis or rebuilds damaged dendrites. But all these effects are courtesy of the drug’s affinity for retinoid X receptors. Enter a totally new idea. Researchers led by Michele Vendruscolo, University of Cambridge, U.K., propose the cancer drug stops Aβ from aggregating into oligomers. The results come from a large research effort in search of therapeutic candidates that target specific points on Aβ’s path to larger species. The collaboration includes Tuomas Knowles and Chris Dobson, also at Cambridge, and Sara Linse of Lund University, Sweden. “This is a very interesting paper that follows from [the authors’] previous work defining the mechanisms of Aβ42 aggregation,” wrote Gary Landreth, Case Western Reserve University, Cleveland Ohio. “This proposed mechanism of bexarotene action is entirely novel.” 

The Alzheimer’s field had hopes for bexarotene, also known as Targretin®, when Landreth first reported striking effects in AD mouse models, but researchers have since had trouble replicating the findings (Feb 2012 news; May 2013 news). Recent clinical data offers mixed results (see Part 1 of this story). 

Meanwhile, the drug’s exact mechanism remains elusive. As a retinoid X receptor (RXR) agonist with a host of genetic effects, it supposedly boosts expression of ABCA1, which helps lipidate ApoE and clear soluble Aβ. Landreth’s group recently reported that it helps clear plaques by enhancing microglial phagocytosis of Aβ (Savage et al., 2015). Other groups variously reported effects on neurogenesis and neuron hyperexcitability (Mounier et al., 2015; Bomben et al., 2014). 

Vendruscolo and colleagues came across bexarotene when collating a library of chemical compounds they believed might interfere with Aβ aggregation based on their structure. Tramiprosate, previously identified as an anti-aggregating molecule, also turned up. Tramiprosate failed in AD clinical trials (Aug 2007 news) but is being rolled out again for a renewed attempt. Vendruscolo and colleagues decided to test whether these compounds could disrupt the formation of Aβ aggregates and, if so, at what point of the process they might do so.

This group developed a kinetics approach to study Aβ42 aggregation (Dec 2009 news). They use a mathematical expression “master equation” that describes theoretical stages, or sub-processes, in aggregation: primary nucleation, where monomers form oligomers; elongation, when monomers add on to existing fibrils; and secondary nucleation, in which fibril surfaces catalyze the formation of more oligomers. Then they fit that equation to data from test tube fibrillization experiments to determine the rates of each sub-process. Using thioflavin T (ThT) fluorescence to monitor fibril formation in real time, they found that primary nucleation drives Aβ oligomerization initially, but after sufficient numbers of aggregates have formed, secondary nucleation becomes the dominant breeding ground for new oligomers (May 2013 news). 

With their knowledge of Aβ fibrillization kinetics and their master equation in hand, the researchers looked for compounds that could perturb specific steps of aggregation. “Differentiating between sub-processes is absolutely crucial for drug development,” Vendruscolo told Alzforum. “Whether you target one or another completely changes the type of therapeutic intervention.” For instance, taking aim at primary nucleation is a great prevention strategy, while targeting proliferation is more therapeutic, he said. Inhibiting certain sub-processes could be counterproductive. “If you target the wrong step, you may increase oligomers and get greater toxicity, despite reducing the total aggregate load,” said Vendruscolo. Molecules that decrease the rate of elongation, for example, would free more monomers to oligomerize.

To put this theory to the test, first author Johnny Habchi and colleagues mixed Aβ, thioflavin T (ThT), and varying concentrations of bexarotene and tramiprosate. Even at high doses, tramiprosate did not inhibit any part of the aggregation process. Previous reports claimed the compound interfered with aggregation (Gervais et al., 2007). 

The data tell a different story for bexarotene. With increasing concentrations of the drug came longer delays in the initial aggregation. However, once enough oligomers had formed, fibrillization took off as usual. The data, and predictions from the group’s theoretical kinetics, suggested bexarotene reduces the rate of primary nucleation, but only weakly affects elongation or secondary nucleation. That data suggest that while the drug might prevent fibrils from forming, it would not dissolve them. 

To test this in an animal model, the researchers exposed Aβ42-expressing nematode worms to increasing concentrations of bexarotene at different developmental stages. When given at the larval stage, before Aβ aggregation had started, the drug prevented deposits from forming and the worms grew up amyloid-free (see image above). However, if worms were exposed at two days old, by which time they were chock full of amyloid, bexarotene did nothing to reduce amyloid. The in vivo data confirmed that the drug prevents primary nucleation.

Together, the results point to an alternate mechanism by which this RXR agonist might protect against Alzheimer’s. The authors have since found several other compounds that more potently delay primary nucleation. If they can be developed into drugs, these could prevent amyloid from forming, they said. Using their systematic approach, they have also found compounds that prevent secondary nucleation. Those could be developed to treat the disease, Vendruscolo said. His ultimate goal is to create a cocktail of compounds that target these two key steps in the aggregation process and develop it as a combined drug.

Does blocking primary nucleation of Aβ explain how bexarotene reduced Aβ and plaques in mice? It is plausible that bexarotene affects Aβ42 nucleation in rodents, Landreth wrote, because the doses Vendruscolo et al. used are comparable to those reached in the mouse brain. However, enhanced phagocytosis better explains the loss of existing plaque, Landreth said, since blocking nucleation would only prevent new deposits from forming.

Jeffrey Cummings, Cleveland Clinic Lou Ruvo Center for Brain Health, Las Vegas, who just completed a Phase 2a clinical trial of bexarotene in AD patients (Cummings et al., 2016), suggested that an effect on primary nucleation could explain the bump in serum Aβ he observed in patients. Nicholas Fitz, University of Pittsburgh, agreed, saying that less primary nucleation could prevent plaques from forming and result in a larger pool of soluble Aβ that could be more easily cleared than aggregates. He and Cummings surmised that bexarotene has multiple mechanisms.—Gwyneth Dickey Zakaib

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References

Therapeutics Citations

1. Bexarotene
2. Alzhemed™

News Citations

1. Upping Brain ApoE, Drug Treats Alzheimer's Mice 10 Feb 2012
2. Bexarotene Revisited: Improves Mouse Memory But No Effect on Plaques 23 May 2013
3. Bexarotene—First Clinical Results Highlight Contradictions 11 Feb 2016
4. FDA Deems U.S. Alzhemed Trial Results Inconclusive 28 Aug 2007
5. Make or Break—Equation of Everything Fibrillar? 18 Dec 2009
6. Aβ Fibrils Drive Oligomer Formation, New Model Suggests 24 May 2013

Paper Citations

1. Savage JC, Jay T, Goduni E, Quigley C, Mariani MM, Malm T, Ransohoff RM, Lamb BT, Landreth GE. Nuclear receptors license phagocytosis by trem2+ myeloid cells in mouse models of Alzheimer's disease. J Neurosci. 2015 Apr 22;35(16):6532-43. PubMed.
2. Mounier A, Georgiev D, Nam KN, Fitz NF, Castranio EL, Wolfe CM, Cronican AA, Schug J, Lefterov I, Koldamova R. Bexarotene-Activated Retinoid X Receptors Regulate Neuronal Differentiation and Dendritic Complexity. J Neurosci. 2015 Aug 26;35(34):11862-76. PubMed.
3. Bomben V, Holth J, Reed J, Cramer P, Landreth G, Noebels J. Bexarotene reduces network excitability in models of Alzheimer's disease and epilepsy. Neurobiol Aging. 2014 Sep;35(9):2091-5. Epub 2014 Apr 2 PubMed.
4. Gervais F, Paquette J, Morissette C, Krzywkowski P, Yu M, Azzi M, Lacombe D, Kong X, Aman A, Laurin J, Szarek WA, Tremblay P. Targeting soluble Abeta peptide with Tramiprosate for the treatment of brain amyloidosis. Neurobiol Aging. 2007 Apr;28(4):537-47. PubMed.
5. Cummings JL, Zhong K, Kinney JW, Heaney C, Moll-Tudla J, Joshi A, Pontecorvo M, Devous M, Tang A, Bena J. Double-blind, placebo-controlled, proof-of-concept trial of bexarotene Xin moderate Alzheimer's disease. Alzheimers Res Ther. 2016 Jan 29;8:4. PubMed.

Further Reading

News

1. United in Confusion: TREM2 Puzzles Researchers in Taos 23 Feb 2015
2. Bexarotene’s Effects Vary by ApoE Genotype, Amyloid Pathology 26 Sep 2014
3. Has ApoE’s Time Come as a Therapeutic Target? 23 May 2014
4. Could Bexarotene Treat Parkinson’s Disease? 18 Oct 2013