3 November 2004. A mouse model that mimics both signature pathologies of Alzheimer disease develops a day-to-day forgetfulness at four months, a young adult age when the animals' brains accumulate the Aβ peptide inside their neurons but don’t yet harbor plaques or tangles. What’s more, a treatment proffered for years by an Israeli scientist appears to reverse this forgetfulness and both pathologies. If these findings hold up, they will stand out from among the more than 900 AD-related presentations at the 34th annual conference of the Society for Neuroscience, held last week in San Diego, California.
The mice in question are Frank LaFerla’s triple transgenics (3xTg-AD), which carry clinical mutations in the human APP, presenilin 1, and tau genes (see Oddo et al., 2003). These mice have generated widespread attention; indeed, LaFerla’s laboratory at University of California, Irvine, has responded to continuing requests from investigators by breeding and shipping the mice to investigators from nearly 20 countries and all across the United States. At the same time, other researchers caution that the model is artificial in its own way, as no humans are known to carry mutations in all three genes. Humans also do not have such high levels of overexpression as do most other AD mouse models.
In San Diego, the LaFerla lab reported their analysis of these mice in regard to lipids, oxidative stress, calcium channels, microglial activation, inflammation, and other aspects in 12 separate presentations. This news summary will focus on three of those dealing with cognition and treatment.
Lauren Billings described her search for a molecular change that marks the earliest cognitive deficit in the mice. She used the Morris water maze, a hippocampal task, and a fear-conditioning task testing function of the amygdala. At two months, the mice learned and remembered normally, indicating that the mice were not born with cognitive impairments, and whatever deficit they developed later was progressive and related to the transgenes. An ongoing longitudinal study has reached the 12-month point to date.
At six months, the 3xTg-AD homozygous mice needed more time to learn the task at hand, but they did learn it. An intriguing clue emerged when Billings broke down the data from this group effect to a trial-by-trial basis. The mice had four training trials per day, spaced apart by 30 seconds. The wild-type controls remember what they’ve learned the day before and therefore start off at a higher performance level every subsequent day of training. The 3xTg-AD also learn from test to test on a given day but by the next day have forgotten and repeat their learning curve from the day before. Compared to previously described learning deficits in AD-related mouse strains, this early result is a seductive one because it echoes a memory problem well known to AD clinicians, where a patient will remember a standard word list for a few minutes, but an hour later has no recollection of it at all.
But is this truly the earliest deficit in this mouse strain? Probably not, the scientists reasoned, because six-month-old homozygous animals showed a deficit during the 1.5-hour time course plus one from day to day, whereas hemizygous animals at this age were defective only in the latter measure. This meant that perhaps younger homozygous animals have only this 24-hour deficit, and indeed the four-month-olds did.
At this age, the researchers can detect only intraneuronal Aβ, not yet plaques or tangles. “This is correlation. We want to be able to say cause,” LaFerla said. The scientists then injected an anti-Aβ antibody (the monoclonal 6E10, which recognizes amino acids 1-17 of human Aβ) into the mice’s third ventricle, and found that it not only clears away intraneuronal pathology from the hippocampus, but also rescues the memory retention deficit. This worked only for the water maze task, however, not for the fear-conditioning task, as the pathology was not cleared in this brain region. This finding may reflect the amygdala’s distance from the injection site, the researchers assume.
“The bottom line is that early cognitive deficits follow closely with intraneuronal Aβ,” LaFerla said. “My personal belief is that the first cognitive deficits in AD are a functional change, not structural. At the point where memory retention first declines, there is not yet loss of synapses, no plaques, no tangles, no dystrophic neurites, no inflammation. Those all come later.”
When asked about the relevance of their model, LaFerla and Salvatore Oddo noted that it did predict accurately some observations of the AN-1792 trial, namely that it failed to remove mature tangles but appeared to reduce soluble tau (unpublished, but see Ferrer et al., 2004). They say this jibes with their recently published data (see ARF related news story), which supports the hypothesis that Aβ accumulation leads to tau pathology in vivo.
Oddo’s talk in San Diego builds on his prior finding that injection of an anti-amyloid antibody clears extra- and intraneuronal Aβ. This again highlights the open question of whether intraneuronal Aβ accumulation contributes to the extracellular plaques (see ARF Live Discussion and ARF Philadelphia news story). To address it, Oddo injected antibody into the mice’s brains once and analyzed the brains not days and weeks later, as in their August paper, but 6, 12, and 18 hours after the shot. Again, he saw that extracellular Aβ disappears first, followed by intraneuronal Aβ. Hours later, the intraneuronal Aβ aggregates first return, then the extracellular ones. This implies that the two pools are connected by a dynamic equilibrium. The finding that intracellular Aβ reappears before extracellular Aβ suggests that intraneuronal accumulation may be a precursor to the plaques, said LaFerla. Other scientists were impressed by this study, but noted that they would like to see follow-up work formally rule out the possibility that the intraneuronal Aβ represents the Aβ sequence within APP.
Last but not least, these data establish a basis on which to test the prowess of potential treatments, and this was the topic of the Antonella Caccamo’s poster. Caccamo injected into the 3xTg-AD mice’s intraperitoneal cavity—every 24 hours over two months, a tiring total of 2,800 times—the compound AF267B. Aficionados of the field may recognize it by its name as one of Abraham Fisher’s. Fisher and his colleagues, at the Israel Institute for Biological Research in Ness Ziona, have synthesized and studied series of small molecules in an effort to prove Fisher’s hypothesis that agonists that are highly selective for M1 muscarinic acetylcholine receptors could treat the symptoms of AD, as well as change its course, (see, for example Fisher et al., 2003 and Fisher et al., 1998). Some muscarinic agonists have been tested for years in vitro and in humans, but none have made it all the way to a useful AD drug. Some early muscarinic agonists have failed in clinical trials. That has made Fisher’s quest seem quixotic to some, despite his insistence that the failure was due to the compound's inadequate M1 selectivity and poor pharmacokinetics. Caccamo put Fisher’s hypothesis to the test, and her data appear to vindicate him. “Everything Abe Fisher has written has come true in our study,” LaFerla said.
Caccamo presented immunocytochemistry data suggesting that AF267B diminished the mice’s plaque pathology, intraneuronal pathology, and tau pathology in cortex and hippocampus. ELISA and Western blots also indicate decreases in soluble and insoluble Aβ formation. The Western blot shows a decrease in C99 (the product of β-secretase cleavage) and an increase in C83 (the APP fragment released by α-secretase cleavage.) Measuring steady-state levels, Caccamo and colleagues found a decrease in BACE, an increase in ADAM-17, and no change in ADAM-10 (see ARF related news story). AF267B also diminished phospho-reactive tau as stained with the antibody AT8. Finally, the compound reversed the memory retention deficit in the water maze.
“This is the first in-vivo evidence for Abe’s prediction of how this compound would shift APP processing and affect tau,” said LaFerla. At the Neuroscience meeting, and also in Neurobiology of Disease this month (see Farias et al., 2004), Fisher and colleagues at the Catholic University of Chile in Santiago laid out a mechanism for how this compound might counteract Aβ toxicity. In short, they propose that AF267B, via activation of the M1 receptor, inhibits the tau kinase GSK3-β and restores a downregulation of the wnt signaling pathway caused by Aβ.
Many questions remain. One fly in the ointment is that AF267B reversed neither the fear-conditioning deficit nor AD pathology in the amygdala. LaFerla hopes that an ongoing collaboration with memory researcher James McGaugh, also at UC Irvine, will shed light on this issue. Moreover, it's not yet clear that AF267B will meet the brain penetration and pharmacological requirements to become an AD drug.
Even so, this early data proposes to show for the first time a small molecule that can cross the blood-brain barrier, is bioavailable, and reverses Aβ and tau pathology as well as some behavioral deficits at doses that cause no adverse effects in mice. A biotechnology company in California has licensed the compound.—Gabrielle Strobel.