11 August 2010. Beyond the tried-and-true, though woefully imperfect lab mouse, Alzheimer disease researchers may have access to a growing repertoire of brainier, brawnier species for their in vivo studies. In particular, vervet and rat models strutted their stuff at the recent International Conference on Alzheimer’s Disease, held 10-15 July at the Hawai’i Convention Center in Honolulu. Cynthia Lemere of Brigham and Women’s Hospital, Boston, reported that Caribbean vervets naturally develop not only pathology but also cognitive decline and the fluid and plasma biomarker changes that mimic human aging and AD. And Terrence Town of Cedars-Sinai Medical Center and the University of California, Los Angeles, presented brand-new data on a transgenic rat that exhibits robust amyloid and tau pathology and a key feature absent from the vast majority of current rodent models—neuron loss. If these data hold up, the new models seem poised to advance AD drug discovery by expanding possibilities for translational research.
Imported from Senegal to the eastern Caribbean island of St. Kitts, the vervets (aka African green monkeys) in Lemere’s study are 96 percent homologous to humans and live some 20 years in the wild, up to 30 in captivity. In an Elan/Wyeth-funded study, aging vervets responded well to immunotherapy with an N-terminal Aβ vaccine; Immunized animals had reduced plaque load and improved memory (Lemere et al., 2004 and ARF related conference story). At this year’s ICAD, Lemere presented behavioral and biomarker data from an ongoing longitudinal study on the vervets, as well as extended pathological data. The Boston researchers collaborate with McGill scientists Roberta Palmour and Frank Ervin at the Behavioral Science Foundation in St. Kitts, who maintain a colony of ~1,000 vervets and conduct all behavioral testing. When an animal in the colony dies, they fix or freeze tissue samples and send them to Boston for pathological and biochemical analyses.
For the longitudinal study that began in 2007, scientists in St. Kitts collect blood twice and CSF three times annually from young (five to 10 years), middle-aged (11-15 years), and old (16-26 years) vervets, 10 females and 10 males per group, and ship the samples to Boston for biomarker analysis. With help from Anne Fagan at Washington School of Medicine, St. Louis, Missouri, Lemere’s group found that in CSF, Aβ42 levels rise as the vervets age, as do scores on an object retrieval test for episodic memory. However, about half of the old animals have dramatic drops in Aβ42 that associate with declining performance on that test, much like what happens in people with preclinical AD. Using a Rules-Based Medicine proteomics assay, the researchers have preliminary data suggesting that plasma levels of several inflammatory proteins, for example, complement C3, IL-1 receptor α, CD40 ligand, and cortisol, may go up with age and correlate with memory loss. Stereological neuronal counts are underway in the basal nucleus of Meynert and hippocampus in aged vervets. Grossly, “it looks likely that some animals show at least some neuron loss,” Lemere wrote in an e-mail to ARF.
Extending previous pathological studies to include 28 vervets (age range 12 to 32 years) to date, “we’ve seen at least some Aβ deposition and vascular amyloid in all animals 17 years and older,” Lemere said. Like in people, more plaques had Aβ42 than Aβ40. The plaques build up in frontal and parietal areas, temporal cortex, with fewer seen in hippocampus. There is great variability among animals; some show surprisingly little plaque deposition even into their twenties. Many of the plaques and vascular amyloid contained pyrogluAβ3-42, a post-translationally modified form of Aβ made by truncating the N-terminus and cyclizing its new end (see ARF related news story).
While activated microglia cozied up to many of the compact plaques, hyperphosphorylated tau did so far less often. “Every once in a while, we’ll see some intracellular tau inclusions—possibly tangle-like but more likely just inclusions. And in three animals, we’ve seen a very rare intracellular staining pattern,” Lemere said. “But it’s just in several neurons, not many neurons per brain.” The researchers picked up some phospho-tau in the monkeys’ cerebrospinal fluid (CSF), as well, “but only rarely in old animals,” Lemere reported. “It’s not something we see in mildly impaired or younger animals at all.”
All told, the data suggest that the vervets may “provide a good model for translational research,” Lemere said, noting that several company researchers who heard the talk have approached her about using the vervets for their preclinical studies.
A Transgenic Rat That Has It All?
A clear disadvantage of vervet studies—the cost of setting up and maintaining colonies—could be mitigated with smaller animals that can still recapitulate the full slate of AD pathological and behavioral features. At an ICAD Hot Topics session, Town argued that his TgF344-AD rat may be the first rodent to fit the bill. This AD model is the result of a collaboration with Robert Cohen and postdoctoral researcher Kavon Rezai-Zadeh at the same institutions. It debuted earlier this spring at a Keystone meeting in Copper Mountain, Colorado (see ARF related conference story), and made a splash in Honolulu with additional data on tau pathology and cognitive decline.
Why create a rat AD model? Mouse models do a good job mimicking cerebral amyloidosis, Town said, but many lack tau pathology and most show no appreciable neuron loss. Furthermore, rats are four to five million years closer evolutionarily to humans and, like people, express all six tau isoforms, Town believes, whereas mice only express three. There is debate about the number of tau isoforms in rats, in part arising from differences in the biochemical protocols used to separate the isoforms on electrophoretic gels; for his part, Town cited a recent paper that he believes supports the presence of all six tau isoforms in the rat (Hanes et al., 2009).
With both transgenes driven by the mouse prion promoter, the TgF344-AD rat makes a triple dose of human amyloid precursor protein with the Swedish mutation (APPswe), and overexpresses exon 9-deleted human presenilin-1 (PS1ΔE9) 13-fold, Town said. Amyloid deposition gets underway around six months of age, and generally precedes tau pathology. Town reported that his team has detected oligomers (i.e., 5-mers), in addition to Aβ monomers, within brains of the transgenic rats. In 16-month-old rats, the researchers found abnormal tau decorating Aβ plaques in the cingulate cortex, as seen by immunogold labeling. Aging rats also racked up increasing amounts of insoluble tau and of the pathogenic tau-associated kinases Cdk5 and GSK3.
It appears these pathological developments could be choking the life out of neurons, or at least correlate with their demise, Town said. Neuron numbers in the hippocampus and cingulate cortex were down 23-38 percent in TgF344-AD rats compared to wild-type controls when determined by blinded manual subfield counting, and as much as 45 percent by stereological counts, Town said. The neurons appear to die by apoptosis in close vicinity of Aβ plaques, as judged by TUNEL and caspase-3 analyses. Furthermore, as shown in behavioral studies not yet complete when Town introduced the TgF344-AD rat at Keystone, learning and memory starts fading by around six months and intensifies through 15-16 months of age. The researchers measured cognition by performance in an open field test and in the Barnes maze.
Because it faithfully reproduces all major AD pathological features, the TgF344-AD rat should become a widely used model for advancing drug discovery, Town said. “The Aβ models are excellent tools, but I’m a bit worried that we haven’t been able to translate any of the therapeutics from mouse models to humans.”—Esther Landhuis.