22 February 2006. This is part 3 of a 5-part series. See also part 1 and part 2.
From Academia: New Leads for Next Generation
Not surprisingly, biomarker research on amyloid plaques and soluble Aβ has a long track record given how central these players have long been to AD research. But are they really the best markers, or merely the oldest, most entrenched ones? And what else is coming down the pike?
One academic perspective on new biomarkers based on soluble Aβ and tau came from Karen Ashe at the University of Minnesota. Ashe, who is one of this year’s recipients of a MetLife award, suggested that novel biomarkers could be found by focusing squarely on the particular forms of Aβ and tau that cause the memory impairment in AD. Ashe laid out recent studies in her lab that attempt to identify those, and her studies on a 12mer called Aβ* is in press at Nature. The Alzforum has recently covered this work in detail; see SfN conference story on Aβ; SfN conference story on tau; and ARF recent news story).
Likewise, Lennart Mucke, of University of California, San Francisco's Gladstone Institute, invited the field to reach beyond traditional readouts toward new, functional markers, especially of synaptic biology. He began by saying that academia fundamentally shares industry’s view of what makes an ideal biomarker. Above all, it must be clinically meaningful, and this particularly has been a knotty problem in AD. New marker candidates are being found in animal studies, and in the future, the field will have to transfer knowledge about them into new radiological imaging agents in humans. In addition, scientists can look for equivalents of mouse markers in human brain, CSF or, ideally, blood and urine.
In AD research, the major pathogenic players are also the biomarkers. Aβ accumulates and forms different types of larger assemblies that then impair neural transmission and reduce expression of activity-dependent markers. For its part, tau accumulates into neurofibrillary tangles but increasingly is also thought to be pathogenic in small, oligomeric forms. For a recent review on these proteins as diagnostic CSF biomarkers, see Andreasen and Blennow, 2005. Studies on thousands of patients, largely in Europe, have established that AD patients have increased CSF tau, particularly forms phosphorylated at specific residues, whereas their CSF Aβ42 tends to be decreased. Phospho-tau cleanly distinguishes AD from normal aging, but when compared with related illnesses such as vascular or frontotemporal dementia, it separates the groups less well. Hence, the current trend in the field has become to combine phospho-tau with MRI hippocampal volumetry and Aβ measurements. (See also Davies presentation.)
Such combined measures still don’t track satisfactorily with cognitive measurements such as MMSE performance. Why is this, Mucke asked? Part of the answer may have to do with the great variety of Aβ and tau assemblies present in the brain. It is unclear exactly which forms a given biomarker assay captures relative to all forms that are present, particularly relative to the forms that do the most damage to the brain at the time of measurement. For example, a total Aβ assay may not reflect oligomers, which may be as pernicious for cognition as plaques. Here, too, new methods of measuring Aβ oligomers are coming on line (Georganopoulou et al., 2005).
Mucke outlined proposed pathways for how AD develops subsequent to APP expression. The question of whether Aβ oligomers, or plaques and dystrophic neurites do the most damage to cognition in AD remains unsettled. Regardless of the answer, therapies reducing Aβ production (i.e., secretase inhibitor drugs) should change biomarkers and neurologic outcome measures in parallel. In practice, many types of therapy targeting one species of Aβ may indirectly draw down other species, as well. But this may not be true for other therapies that specifically target a more downstream segment of the pathway such as amyloid deposition, Mucke noted. Examples would be plaque-busters, or drugs that selectively inhibit fibril formation but not oligomerization. To the extent that they increase pathogenic oligomer levels, such drugs might actually worsen cognitive deficits, and in this case, using amyloid plaques as the biomarker of choice could create the problem Lieberburg outlined in part 2 of this series, where the biomarker responds to the drug but is clinically irrelevant. On the flip side, a drug that moves oligomers into deposition might improve neurologic deficits even while increasing amyloid load. Therefore, drug studies could benefit from monitoring cognition-related biomarkers in addition to standard measures of plaque and amyloid load, Mucke suggested. There was widespread agreement that for the field to move past these questions, it will be critical to develop more sophisticated ways of measuring Aβ that distinguish its different forms during the aggregation process.
This is more than speculation, as animal model data have begun to separate amyloid deposition from cognition—at least what passes for cognition in mouse strains. For example, consider new mouse models of mutant APP made by Irene Cheng in Mucke’s lab. A related ARF conference story already summarizes this work. In brief, the study allows comparison of a particularly fibrillogenic mutant form of Aβ to wild-type Aβ in otherwise similar transgenic mouse strains. The new lines develop amyloid plaques and associated neuritic dystrophy earlier than do the J20 comparison line, but this rapid deposition does not track with neural function. On the contrary, the mice with the fewest plaques performed more poorly on the Morris water maze than did lines whose brains were laden with plaques. The scientists interpret this to mean that the fibrillogenic mutation diminishes the pool of bioactive oligomers even as it speeds up fibrillization, suggesting that plaques are relatively protective compared to oligomers.
Mucke then moved on to describe new studies of other biomarkers that correlate more closely with cognitive impairment than does Aβ. His lab explores markers whose levels depend on excitatory synaptic transmission, and found that these tend to be depleted in relevant brain areas in the presence of elevated Aβ. Researchers including Bloom and Cole have shown before that dendrites of hippocampal granule cells are susceptible to the effects of Aβ, and Mucke’s group reported a drop in the synaptic calcium-binding protein calbindin in dentate gyrus of APP transgenic mice and in people with AD (see ARF related news story). This is not due to neuronal loss; rather, the neurons were still there, but their synapses were altered. Calbindin levels correlated well with water maze performance in mice, and with cognitive scores in humans.
It’s not just calbindin, either. Other activity-dependent markers change in APP-transgenic mice, especially in the dentate gyrus subregion of their hippocampus. An example is the immediate early gene arc, which acts locally at activated synapses and helps maintain LTP and consolidate memories. Experiments using an enriched environment showed that APP-transgenic mice are dramatically less able to induce this marker than are wild-type mice (Palop et al., 2005). Prior mRNA measurements in hippocampus done in David Morgan’s laboratory have suggested that already (Dickey et al., 2003). Arc interacts with many other synaptic proteins. The pathways of synaptic molecular biology, and the effect of Aβ and tau on them, deserve more study in the search for biomarkers linked to cognition. One of arc’s upstream regulators of interest is the extracellular matrix protein reelin, which functions in LTP and dendritic reorganization. A separate mediator of Aβ’s synaptic toxicity is the kinase Fyn (Chin et al., 2005). In this context, tau only heightens its notoriety, as it is necessary for APP-transgenic mice to develop behavioral deficits (see ARF conference story).
In summary, recent work on animal models suggest that plaques remain an important outcome measure for secretase inhibitors and for Aβ removal, that is, immunotherapy. Yet, where the goal is to assess cognition, Mucke recommends that researchers also develop measures of synaptic activity. For that, new markers established in mouse studies should be moved into human imaging, where they could complement existing measures of regional volume loss, cerebral glucose utilization, and amyloid imaging.
Greg Cole of UCLA continued the theme of searching for new biomarkers in the withering synapses of AD. Cole’s prior work on testing different therapeutic approaches in transgenic mice has identified new candidates, some of which are known to play a role in mental retardation. It has also identified dietary ways of influencing the underlying pathways. Cole’s recent work points toward the interplay of Aβ oligomers and proteins involved in the cytoskeletal rearrangements of synapse and spine formation.
Broadly speaking, the conventional view that neuron loss drives synapse loss in AD is gradually yielding to one where neuron loss is not the initial problem. The real question has become what is driving cognitive deficits, and the answer to it may yield not only new markers of cognition, but also more treatable targets, Cole noted.
What biomarkers of cognitive function already exist? Synaptophysin is the best-known one, and its levels closely track tangle formation. Paul Coleman and others have shown a tight and compelling correlation between tangle formation and synaptophysin loss, where human tangle-bearing neurons have a large reduction in synaptophysin message. This marker is less useful in AD transgenic mice Cole uses, because they do not show marked synaptophysin loss. The mice do, however, have cognitive deficits, and they also have in common with AD that both show loss of spines, shrinking dendritic arbors, and loss of dendritic area (for more on this, see section on Bloom, part 5). One way of approaching the molecular biology of cognition is to look at mental retardation genes, said Cole, because different inherited forms of mental retardation share spine defects that are similar to those seen in AD and Down syndrome. Cole is testing the hypothesis that Aβ aggregates cause these dendritic spine defects, and that solving the cognitive problem would require repair of the spine defects.
Aβ aggregates likely affect these processes. They are known to induce rapid LTP deficits, probably by mechanisms that include microglial activation and down-regulation of components NMDA and AMPA receptors (see ARF related news story). All this is consistent with the broader idea that AD entails a postsynaptic attack on excitatory spines, Cole said (see Bloom; Moolman et al., 2004; Spires et al., 2005; Dickey et al., 2004). Cole is particularly interested in the synaptic protein drebrin, an actin-binding protein in spines that occurs primarily in cells containing PSD95. Both proteins are lost in AD brain and in APP transgenic brains.
Cole believes that diet and oxidative stress modulate the proposed attack on spines. A diet that depletes the omega-3 fatty acid docosahexaenoic acid (DHA) exacerbates it (Calon et al., 2004; Calon et al., 2005), and dietary DHA deficiency in transgenic mice accentuates oxidative damage and correlates with the loss of a panel of synaptic markers including the NMDA receptor subunits NR1 and NR2, as well as CamKII. The idea that people can develop cognitive impairment from deficits in an NMDA receptor component has support in mouse models. In particular, Cole suggests that an interaction between the transgene (or in AD, Aβ oligomers) and diet leads to the oxidization of DHA’s double bonds in neurons and synapses. Part of the mechanism could be that DHA availability influences the activation of AKT, the nexus of a prominent survival pathway in neurons (Akbar et al., 2005). In theory, AKT activators could be helpful, though in practice they would have to be weighed carefully against the established role of AKT in fueling some cancers.
Incidentally, DHA is being added to some brands of infant formula to support brain development. The brains of infants who have not yet developed elaborate dendritic arbors look strikingly similar in PET scans of glucose utilization to those of late-stage AD patients, who have lost them, Cole noted.
To search for pathways that mediate Aβ’s role in synapse formation, Cole’s group, in collaboration with Sally Frautschy’s, performed a microarray analysis of their transgenic mice. The researchers measured expression differences in a panel of previously identified mental retardation genes between animals on a DHA-rich and a DHA-depleting diet. They focused on PAK kinases because these enzymes control actin dynamics in dendritic spines and appear to protect against loss of drebrin, Cole said. Other groups have shown that PAK inhibition by itself can cause cognitive deficits. APP-transgenic mice on the unhealthy diet lost PAK message, and their remaining PAK protein clustered around amyloid plaques instead of being distributed near synapses, where it normally occurs. AD brains showed severe depletion of soluble PAK and similar changes in PAK staining, Cole reported. Furthermore, in a culture model of Aβ oligomers/ADDL colocalization with NMDA receptor and PSD95-containing sites, PAK also changes its normal diffuse staining around synapses to a clustered pattern two hours after oligomers are added and taken up into the neurons. This suggests a PAK translocation has occurred in response to the Aβ oligomers. Drebrin staining went down in tandem with PAK, but adding intact PAK protected the cultures against drebrin loss. Back in vivo, infusing anti-Aβ antibody into APP-transgenic mice increased PAK just as levels of a 12mer form of Aβ decreased, Cole said. (This is the same 12mer Ashe studies, article in press at Nature.) In short, Cole is implicating Aβ aggregates in a PAK pathway defect.
It is vexing that the first step of any proposed pathway leading from Aβ via synaptogenesis proteins to spine loss is still a mystery, Cole noted. This is, of course, the identity of the Aβ receptor; integrins are sometimes mentioned as a candidate and some labs have hinted of having found a receptor but are staying mum until formal publication. Even so, this work suggests common pathways between developmental and age-related cognitive impairment. In the former, mutations in mental retardation genes are often the cause. In the latter, Aβ would induce loss of PAK and drebrin, and that, in turn, would impair formation of the protein complexes that are needed for remodeling the actin cytoskeleton in dendrites and spines as the brain tries to form new synapses. Part of Cole’s work was recently published (see ARF PAK/p21 news story). Cole suggested PAK as a new biomarker that might be amenable to treatment. Experimental treatment with curcumin lowered Aβ levels and reversed PAK and drebrin deficits in APP transgenic mice. Insulin also affects PAK levels. In this context, a growing literature describing overlaps between insulin dysregulation and dementia is of interest, most recently an MRI study documenting that people with type 1 diabetes have subtle reductions in gray matter in brain areas responsible for memory, language processing, and attention (Musen et al., 2006). —Gabrielle Strobel.
See also part 1 and part 2.