5 April 2010. Common belief has held that in Alzheimer disease, buildup of neurofibrillary tangles within neurons switches on caspases that eventually kill the cells. However, an analysis published March 31 in Nature provides evidence for the reverse—namely, that caspase activation may precede tangle formation. Using multiphoton imaging to visualize tangles and activated caspases in real time in the brains of tau-overexpressing mice, researchers led by Brad Hyman at Massachusetts General Hospital, Charlestown, propose a model where caspase activation occurs early, and forthcoming tangles mark, rather than cause, neurodegeneration.
Postmortem studies in tau transgenic mice and AD patients have found that tangles map to brain regions littered with dead (Gómez-Isla et al., 1997; Ramsden et al., 2005) or misshapen (Augustinack et al., 2002) neurons, and that cleaved tau colocalizes with activated caspases and other apoptotic markers (Ramalho et al., 2008; Guo et al., 2004; Rohn et al., 2001). These observations have helped fuel the notion that tangles cause neurodegeneration in AD and other tau-related dementias.
But when Hyman and colleagues began using multiphoton imaging to monitor tau pathogenesis in vivo in Tg4510 mutant human tau transgenic mice, they were surprised to find that tangle-bearing neurons with activated caspases did not die immediately (Spires-Jones et al., 2008 and ARF related news story). In a later analysis, these cells hung around for at least a day after initial imaging (de Calignon et al., 2009), raising the possibility that tau aggregation, even caspase activation, did not spell instant neuronal death in this tauopathy model.
In the current study, lead author Alix de Calignon and colleagues explored these issues further in the Tg4510 model, and in wild-type mice injected intracranially with tau-expressing viruses. Using multiphoton imaging through a cranial window, the researchers detected tangles by staining with thioflavin S, and caspase activation by applying fluorescent indicators that bind activated enzyme. They monitored more than 500 tangle-positive neurons in four seven- to nine-month-old Tg4510 mice (which develop tangles and neuronal loss by seven months) and found they stayed alive through two to five days of observation, reinforcing what they saw previously over shorter timeframes. Consistent with previous studies by the Hyman lab (Spires-Jones et al., 2008), very few neurons (i.e., less than 1 percent) had active caspases, but the vast majority (>87 percent) of those also had tangles. Exceedingly few neurons were caspase-positive without tangles.
Focusing on this rare subclass—of which they found only 22 neurons over 24 imaging sites in three mice—the researchers discovered that 20 (or 91 percent) of the cells formed a new tangle by the time they were imaged the next day (see image below). By contrast, less than 2 percent of caspase-negative neurons formed a new tangle within a day’s time. The authors conclude from these data that tangles can form quickly—in under 24 hours—and that caspases are activated before tangles develop.
Caspases First, Then Tangles
Thioflavin S (green) and caspase indicator (red) were applied on the surface of the brain of Tg4510 mutant human tau transgenic mice. Some tangle-free cells showed caspase activation upon initial imaging (time 0), and most formed a new tangle within a day (time 1). Image credit: Alix de Calignon
Curiously, while virtually all new tangles formed in neurons with caspase activity, the researchers also found that more than 90 percent of tangle-bearing neurons were caspase-negative. This suggested that tangle-containing cells “survive the initial caspase attack and become caspase-negative,” the authors write.
What, then, is responsible for turning on the caspases? Hyman and colleagues figured it might be soluble mutant tau, given that they could not find a single caspase-positive neuron in six wild-type mice or two amyloid precursor protein (APP)-overexpressing mice. To test whether soluble mutant tau was the culprit, they took advantage of the doxycycline-controlled transgene expression of the Tg4510 mice. The researchers monitored for tangles and caspase activation in seven- to eight-month-old Tg4510 and wild-type mice at baseline, and after two or six weeks of doxycycline treatment to block mutant tau expression. Over the six weeks, caspase activation dropped ~20-fold in tangle-bearing neurons, suggesting to the authors that soluble tau molecules are upstream of caspase activation.
Furthermore, they showed that caspase activation colocalized with a truncated form of tau generated by caspase cleavage at aspartate 421 (D421). They did this by imaging the Tg4510 mice in vivo using a pan-caspase indicator, then sacrificing the animals and analyzing their brains for D421-cleaved tau (as measured by an antibody specific for this neo-epitope).
To confirm this in another model, de Calignon and colleagues injected wild-type mice intracranially with viruses carrying wild-type tau, creating a scenario that more closely resembles sporadic AD. In these tau-overexpressing mice, the neurons containing activated caspases also had caspase-cleaved tau. This colocalization of caspase activation and D421-cleaved tau also occurred in older hTau transgenic mice that express wild-type human tau.
The scientists went on to show that this cleaved form of tau could seed aggregation in vivo. Using viruses, they introduced D421-truncated tau into wild-type mice and found that more than a third of neurons expressing this cleaved form developed pathogenic conformations of tau (as measured by Alz50 conformation-specific antibodies). The abnormal Alz50-positive structures also contained endogenous tau. This suggests that truncated tau acts like a loss-of-function mutant, recruiting endogenous tau into aggregates and thereby removing tau from stabilizing microtubules and preventing it performing its normal functions.
In a recent study, Gail Johnson, University of Rochester, New York, and colleagues reported another ill effect of caspase-cleaved tau—compromised mitochondrial function. (Quintanilla et al., 2009). The current paper “provides strong evidence that this form of tau occurs in an AD paradigm, and we can think of it as causing toxic changes to occur,” she said in an interview with ARF. “Everybody's been focusing almost exclusively on tau phosphorylation, but this indicates that caspase cleavage may be a lot earlier and more significant than we realize.” In human disease, Johnson suggested, “You may have cycles of slight caspase activation throughout the life of neurons, until the load of cleaved tau becomes too much for the cell to handle.”
Johnson and others have some concerns. In the author’s proposed model, large amounts of tau accumulate inappropriately in neuronal cell bodies, where they activate caspases that cleave tau to spur tangle formation, at which point caspase activity tapers off and neurons survive in a low-functioning state despite the presence of tangles. This model only describes what could happen when tau is overexpressed and does not address what happens in human disease, noted Jürgen Götz, University of Sydney, Australia, in an e-mail to ARF. Johnson agreed, suggesting that there may be upstream events that cause tau to accumulate in the cell bodies, where they can activate and be cleaved by caspases.
Furthermore, Götz suggested that biochemical evidence is needed to confirm that soluble tau rather than tangles is the trigger for neurodegeneration. Some cells judged to be tangle-free may actually contain fibrillar tau that has not yet reached a level detectable by thioflavin S, he wrote. “Electron microscopy or Western blotting of sarkosyl or formic acid-extracted brain tissue would be helpful.” (See full comment below.)
Others worried that the fluorescent indicator used to stain activated caspases has skewed conclusions because it functions as a pan-caspase inhibitor. The authors said they were aware of this and did a control experiment, which showed that the FLICA indicator wore off quickly relative to the daily imaging they did. “We are confident that even though FLICA covalently binds the active site of caspases, it does not inhibit caspase activity for an extended period in the conditions we used,” de Calignon wrote in an e-mail to ARF (see full comment below). She noted that none of the paper’s reviewers raised concern about the caspase indicator.
All told, the study “really sets up the field and says okay, we have to stop just looking at aggregates,” Johnson said. “It provides strong evidence that neurofibrillary tangles are not toxic. Cells are long-lived after formation of tangles.” Meanwhile, at least several companies are developing drugs that reduce tau filaments. At the 2008 International Conference on Alzheimer’s Disease in Chicago, TauRx Therapeutics, Ltd., a Scottish-Singaporean biotech company, presented Phase 2 data on a drug that interferes with tau aggregation by acting on self-aggregating cleaved tau fragments (see ARF related conference story).—Esther Landhuis.
de Calignon A, Fox LM, Pitstick R, Carlson GA, Bacskai BJ, Spires-Jones TL, Hyman BT. Caspase activation precedes and leads to tangles. Nature. 31 March 2010. Abstract