20 February 2009. At the Keystone Symposium, Neurodegenerative Diseases: New Molecular Mechanisms on Wednesday, debate on the last plenary talk continued around the dinner table and well into the poster session. Marc Tessier-Lavigne, Genentech Inc., San Francisco, California, described a novel role for APP in neurodegeneration, a role that appears completely independent of Aβ toxicity and that might finally explain why only certain neurons bear the brunt of Alzheimer disease pathology. Tessier-Lavigne reported that the extracellular domain of APP serves as a ligand for death receptor 6 (DR6), an orphan member of the tumor necrosis factor receptor superfamily. He showed that when the N-terminus of APP (N-APP) binds DR6, it sets off an apoptotic cascade in embryonic spinal neurons that targets both axons and cell bodies. The work indicates that N-APP has a role in axonal pruning and neural cell death in development, but it also raises the possibility that similar events occur in mature neurons in the brain. “Finding that an N-terminal fragment of APP is a ligand for DR6 came as a complete surprise, and finding that APP is involved in a self-destruction mechanism like this immediately suggested that perhaps it could contribute to Alzheimer’s disease,” said Tessier-Lavigne in an interview with this reporter after the talk. The study was coincidentally published in Wednesday’s Nature.
“This is very intriguing. I think the story is very convincing because there is a lot of very complementary data, biochemical, cell biological, etc., and I do not doubt that it is relevant for APP biology,” said Bart De Strooper, University of Leuven, Belgium, who was at the meeting. He was not involved in the study. “Of course, when you have this type of data I think it is fair to ask how relevant it is for Alzheimer’s disease. I think it is too early to say if it is equivalent to the amyloid hypothesis, for example, because there is no human or clinical data,” he cautioned.
Tessier-Lavigne and colleagues identified the N-APP/DR6 interaction when studying neuronal development. The embryo is a tough environment for new neurons with many more being formed than typically needed. Those that do not make proper connections are eventually weeded out by a process that involves axonal pruning and cell death. First author Anatoly Nikolaev and colleagues found that DR6 plays a key role in this process, kicking off an apoptotic pathway in commissural, motor, and sensory neurons that depends on activation of caspase 6 in axons and caspase 3 in the soma only. Nikolaev and colleagues mimicked this axon degeneration by withdrawing trophic support (such as nerve growth factor, or NGF) from neurons in culture, but if they blocked DR6 at the same time—with an antibody, by knocking it down with RNAi, or by genetic knockout—they were able to prevent axonopathy and cell death. Collaborating with Todd McLaughlin and Dennis O’Leary at the Salk Institute, they also showed that DR6 regulates neuron death and axonal pruning not just in cell culture, but also in vivo in a mouse model.
APP entered the picture when Tessier-Lavigne and colleagues considered what might activate DR6. The protein is a cell surface receptor with no known ligands. In fact, the researchers were not even sure if a ligand was necessary for its activation, but when they incubated neurons with a soluble DR6 ectodomain construct, it prevented degeneration following trophic factor withdrawal. That suggested that a ligand, mopped up by the soluble DR6 protein, was necessary for the process. In support of this, the researchers found that the DR6-AP—the DR6 ectodomain bound to alkaline phosphatase to act as a reporter—detected proteins (100 kDa and 35 kDa) in conditioned medium following NGF withdrawal suggesting that those proteins may be the DR6 ligands that promote axon degeneration.
The researchers took a leap of faith when they decided to immediately focus on APP as a potential ligand. Tessier-Lavigne explained that in many respects APP fit the bill, as it is shed from the cell surface, it is tied to neurodegeneration already through AD, and it is highly expressed in developing neurons. In initial experiments, the researchers found that DR6-AP bound to APP on the surface of COS-1 cells. A polyclonal antibody to the N-terminal of APP also recognized the same 100 and 35 kDa proteins that turn up in conditioned medium after trophic deprivation. An antibody to the C-terminal of sAPPPβ, which is released upon BACE cleavage, also detected the 100 kDa protein in the conditioned medium, and also a 55 kDa protein. The antibody binding patterns suggest that after trophic withdrawal, APP is cleaved by BACE to yield the ~100 kDa sAPPβ, which is further cleaved, by an unknown protease, to yield the 55 and 35 kDa fragments. Tessier-Lavigne said that it is not clear whether this second cleavage is necessary for activation of the DR6 pathway.
A range of additional experiments supports the idea that APP sets axons off on a suicidal slippery slope. Degeneration of sensory neurons by trophic withdrawal is blocked by antibodies to N-APP and by knocking down APP by RNAi. BACE inhibitors also blocked the degeneration in cultured neurons, but it could be restored by adding a purified N-terminal fragment of APP (amino acids 1-286). Finally, the affinity of the N-terminus of APP for DR6 is very high (EC50 for binding is around 4.5 nM) and the interaction seems fairly specific since the researchers found that N-APP only reacts with one of the nine other members of the TNF receptor superfamily they tested, p75, and at 60-fold lower affinity.
Do these data suggest an entirely new model for neurodegeneration in AD? “What it does is it opens up additional possibilities for how APP could be involved in the pathological process and therefore opens up new potential therapeutic targets as well,” said Tessier-Lavigne in a post-talk interview. The data could, for example, explain why specific neurons are targeted in the disease despite widespread expression of APP in the CNS. Tessier-Lavigne showed that DR6 expression in the mature brain is enriched in sites of known AD pathology, including the hippocampus and forebrain cholinergic neurons, suggesting that under certain conditions, shedding of the APP ectodomain might trigger a self-destruct pathway that contributes to neurodegeneration in those very same neurons.
There are other facets of the disease that this model might have more difficulty explaining, however. David Holtzman, Washington University, St. Louis, Missouri, noted during questions that there are familial AD mutations that occur in the Aβ region of APP and that it would be hard to reconcile how they could fit into this model. Tessier-Lavigne agreed that he needs to determine whether and how such mutations might tie in to his mechanism. He also pointed out that the model is not mutually exclusive of the amyloid hypothesis, and could even be complementary. His lab is in the process of crossing DR6 knockout mice with AD mouse models to see what impact the death receptor pathway has on pathology in those systems.
In a post-talk interview, Chris Link, University of Colorado, Boulder, also raised the issue of tau pathology and the formation of neurofibrillary tangles (NFTs). “I think in some people’s view, including mine, Alzheimer’s is ultimately a tauopathy, and it is not clear how this model leads to tau hyperphosphorylation,” he said. Tessier-Lavigne suggested that caspase 6 activation, which occurs downstream of N-APP/DR6 binding, might tie APP to tau pathology. He brought up that work from Andrea LeBlanc’s lab, for example, shows that caspase 6 and tau fragments generated by the protease are elevated in aging and in mild cognitive impairment (see Albrecht et al., 2007). “It is possible that [caspase 6 activation] could lead to tau aggregation because there is good evidence that tau fragments play a role, and phosphorylation could be secondary to fragmentation,” said Link. “The issue is that although you see NFTs, and certainly tau hyperphosphorylation in neurons, they still look like neurons. If they really had degenerated then they would be gone.”
Also, in a News & Views written for Nature, Donald Nicholson, Merck Research Laboratories, Rahway, New Jersey, noted that caspase 6 can also cleave APP in almost the same location as BACE, raising the possibility that the caspase feeds back to generate more N-APP, thereby amplifying the apoptotic process or spreading it to other cells. “Such potential secondary effects are hard to ignore, particularly because they might be relevant to Alzheimer’s disease,” writes Nicholson.
Obviously, there is much more work to do to corroborate these results and to explore the mechanism in greater detail. In addition to mouse model work, Tessier-Lavigne said he wants to look for evidence of activation of the DR6 pathway in the adult human brain and particularly in the AD brain. “Since we have additional players in a biochemical pathway, we would like to know if there are any mutations associated with them that represent risk factors in Alzheimer’s disease,” he added. In fact, DR6 is located very close to a potential susceptibility locus on chromosome 6. “At the same time we are trying to develop potential therapeutic candidates to interfere with steps in this pathway and to use them in preclinical models. If that looks promising, we’d obviously consider moving toward the clinic.”—Tom Fagan.
Nikolaev A, McLaughlin T, O'Leary DD, Tessier-Lavigne M. APP binds DR6 to trigger axon pruning and neuron death via distinct caspases. Nature. 2009 Feb 19;457(7232):981-9. Abstract