Robert Balaban opened the discussion by observing that heart disease and AD research share certain characteristics, including a weak familial linkage, poor penetrance of some of the known risk factors, and a poor understanding of the interplay between environmental and behavioral factors with gene-gene interactions. He challenged the room by stating that the basic cellular pathology mechanism in AD remains unknown. He predicted that this undiscovered mechanism will be specific to certain cells and that it will be cataclysmic because neurons are disappearing, two characteristics that may make therapy development easier. A human pathology mechanism urgently needs clarification in order to validate AD animal models for screening and to develop biomarkers, readouts for cell-based assays, and markers for imaging techniques. Overall, Balaban said, a rational scheme for therapy development does not exist. He recommended a concerted effort to unravel mechanistic pathways.

This introduction provoked debate about how much of the etiology can be explained by elevated Aβ, and whether the underlying signaling cascades must be known before therapies can be developed. Selkoe argued that a complete mechanism of elevated cholesterol had not been worked out prior to the development of statin drugs, and that it was not necessary because LDL production was clearly connected to atherosclerosis. Likewise, elevated Aβ is strongly linked to the AD endpoint, therefore a step-by-step understanding of how accumulated Aβ damages neurons need not be the priority at this point.

Lansbury argued that the pathologic pathways may be manifold and complex. Redundant pathways are at work in the neuron's slow death, and interfering with one would not be effective. Sorting them out will take many years and is not required for testing the hypothesis that blocking the inducing event-Aβ accumulation and fibrillization-will be therapeutic. Lansbury favored embarking on high-throughput screening approaches now to save time.

Others disagreed, saying that understanding the underlying pathways, especially those regulating the cell cycle, inflammatory cascades, disrupted cell signaling, mitochondrial involvement in apoptosis, metabolic failure, and potential autoimmunity was a prerequisite to identifying novel biomarkers and new therapy strategies.

Everyone agreed these pathways ought to be unraveled, but participants disagreed on what should be top priority now. Some argued for hypothesis-driven research into mechanisms to generate biomarkers and better mouse models, others prefer investing in high-throughput screening to generate lead compounds, targets, and novel hypotheses.

On animal models, Jaenisch said that the genetics employed for current AD mice are outdated and could be much improved. Mayeux defended current models, saying they simulate aspects of the disease and enable testing of the basic premise that removing amyloid from the brain improves symptoms. Balaban countered that the pathology seen in mice may not be the real pathology occurring in AD.

Selkoe said synaptic loss deserves more study, rather than loss of cell bodies, because the synapses are key functionally and their loss may precede that of cell bodies. Synaptophysin changes, electrophysiological changes including LTP maintenance and ESP alterations, all occur in mouse models of disease even in the absence of plaques or massive neuronal loss. To this extent, mouse models of elevated Aβ correlate with toxicity.

He said data on Aβ suggest it is not directly toxic to neurons but precedes neuronal injury. Presenilin and AβPP mutations lead to elevated Aβ, as seen in plasma and Down syndrome. No one knows exactly how Aβ damages neurons. Myriad in-vitro studies suggesting its toxicity are inadequate. Animal models show that elevated Aβ causes synaptophysin loss and changes in electrophysiological properties, but that does not support the leap that Aβ is toxic to all neurons lost in AD.

Goate and others said genetics clearly points to AβPP processing abnormalities as key to disease in those cases. Wang said that while genetics clearly points to importance of amyloid, Aβ levels are not elevated in serum early. Therefore other molecular changes must be occurring prior to onset of symptoms, and identifying these other proteins is a priority.

Lo said the key knowledge gap that is making AD difficult for his company to approach is the lack of proteins on which to base assays. Most medium to high-throughput systems are based on a cellular readout. A readout in whole animals is ideal but throughput suffers, so his company developed a brain slice assay to keep the context of cells temporarily intact. In AD he does not know what to look for, Lo said. He needs a proxy that represents some state of progression of the pathology at the cellular level. David Sabatini agreed that reliable markers at any point of the long pathogenic process would accelerate the identification of targets, even before a comprehensive mechanism is worked out. Some of these test points are going to occur years before there is anything to image.

How do we get those markers? Goate said that genetics is trying to uncover them, but a total of six genetics labs competing to pin down the same few candidate genes is too small an effort.

Balaban said FAD genetics are fine but sporadic disease has no mutations. Normal AβPP processing contributes to disease if a different genetic defect or environmental condition leads to downstream sensitization in vulnerable neurons. The lack of complete penetrance of overexpressors and the fact that people without Aβ overproduction get AD, point to other gene-gene interactions and environmental interactions in downstream pathology. All agreed that an explanation for the differential vulnerability of certain neuronal populations is a priority.

Selkoe said it is clear that amyloid accumulation is toxic in other diseases, as well, not how it is but that it is. He said the present discussion follows behind what has been found in 30 years of research on these other disorders, as some of them are treated successfully by inhibiting amyloid production. Rather than focus on holes in the amyloid hypothesis, he urges discussion of better ways to block Aβ?.

Jaenisch asked whether neurons in AD die because of intrinsic problems or influences from its environment, a question that neuronal transplantation studies as developed in embryology could address. Others reply that this was an early question in the AD field, subsequent studies have pointed to extrinsic defects. This issue sparked a discussion of inadequate data in AD on the effects of tissue-specific expression of transgenes. Heywood said that in ALS, the SOD mouse develops disease only if the transgene is expressed in spleen and liver; its expression in neurons and/or astrocytes alone is not sufficient to cause disease.

Coleman, Hyman, and Davies pointed to the hierarchical layers of vulnerability of brain areas as a knowledge gap that can be addressed. Neuronal loss begins in layer 2 of entorhinal cortex and then progresses through the brain in a fairly predictable anatomical sequence. Why? A key priority is to describe what distinguishes the affected cells from the unaffected cells. Nobody has really exploited this opportunity.

Jaenisch said Rett syndrome may be relevant to this question because the protein and molecular mechanism at play acts as a general suppressor of transcription in every tissue, every cell, without any specificity whatsoever. Yet the Rett phenotype is extremely specific. So the question is: is there something especially sensitive in those neurons affected in AD that defines their response to a less specific insult?

The question of apoptosis in AD was discussed. In in-vitro, in-vivo, development, and disease models, cell death always occurs over short periods of time. In AD, cell death seems to occur over decades in an individual neuron. What is the reason for this? Coleman said array studies show that the neuron-postmitotic and designed to last a lifetime-mounts defensive mechanisms. Synapse loss and neurite shrinkage is one such mechanism, "moving troops back from outposts." Many forms of defense prolong the neuron's path to death, and understanding those and devising ways to boost them can lead to therapy.

Inflammation was mentioned as a protective mechanism. DiStefano said that generally, NFkb activation in the nervous system is considered protective, whereas stimulation of caspases is detrimental. He sees in many experimental systems a balance between these two currents, and immune-type or inflammatory functions appear to dictate the balance. They act as life/death checkpoints and may explain why it takes so long for neurons to die. Crudely withdrawing trophic support in vitro causes neurons to die within 24 hours, but in vivo, the assaults are more subtle and protective mechanisms are in place.

DiStefano said activated microglia and astrocytes are the source of some of these immune-like functions and deserve more attention. Are they protective or damaging? Others object to using the term inflammation, because AD does not feature a classic immune response involving peripheral lymphocytes and macrophages.

On the question of normal aging versus AD, all agree that AD is not just accelerated aging. This has become a fringe view. Hyman said his data clearly prove that no massive neuronal loss occurs in normal aging but does in AD. Coleman elaborates that the pattern of cell loss distinguishes AD from normal aging. In CA1 of hippocampus, Coleman found massive neuronal loss in AD but no loss in normal aging, whereas in subiculum, neuron loss is similar in AD and normal aging.

Balaban closed by saying that the current trials of secretase inhibitors and Aβ immunotherapies may fail and that the meeting's charge is to define a research strategy for identifying other Achilles' heels to go after while these trials take place. On that, there is fuzziness beyond amyloid.

All agree Aβ should be pursued therapeutically. All agree a better understanding of underlying pathways would make it possible to identify other sensitive, non-redundant points in the interplay of signaling cascades that may provide new targets. All agree that uncovering the molecular pathology and unbiased screening ought to be pursued in parallel.

These Scientific Priorities Drew Some Consensus:

  • Identify additional genetic and environmental risk factors. None known for 50 percent of AD cases.
  • Develop readouts for use in medium-throughput cell-based and slice assays.
  • Focus effort on understanding differential vulnerability of neuronal cell types, even single cells within a given area. Begin by characterizing more fully the difference in surface markers expressed on most versus least susceptible neurons. Wang says Mass Spec is able to filter out noise. Do a systematic study on both mRNA and protein.
  • Test decisively whether an autoimmune response is at play in AD.
  • Define protective versus damaging aspects of the inflammatory mechanisms at play; define the pathways of microglia and astrocyte activation and their role in neuronal death/survival.
  • Refine animal models to more faithfully mimic AD. Limitations are not inherent to species; rather, current models miss a genetic aspect. For example, make mice transgenic for (micro)glial receptors to mimic induction of inflammation.
  • Bring high-throughput proteomic screening (e.g. in cell-based assays analyzed by mass spectrometry) to bear on AD research.
  • Develop better psychometric instruments.


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