Hyman and colleagues use a powerful in vivo multiphoton imaging approach through craniotomy to monitor the sequence of tau pathology in the inducible tau transgenic mouse model Tg4510, and in mice intracranially injected with tau-expressing AAV (adeno-associated viruses). They come up with the conclusion that caspase activation precedes and leads to neurofibrillary tangles. The data would even suggest that tangles are almost something like a salvage pathway.

The authors also conclude, based on a combination of staining methods, that tangles develop within a day’s time, which is highly reminiscent of their earlier findings, also published in Nature, that amyloid plaques develop within the same time frame (Meyer-Luehmann et al., 2008). The question is, What happens in human disease? Interestingly, in the model presented in the supplement (Supplementary Figure 6), the authors present a “Tangle formation hypothesis” that would operate in tau-overexpressing mice, but not necessarily in humans. Specifically, the authors state that...  Read more

Hyman and colleagues use a powerful in vivo multiphoton imaging approach through craniotomy to monitor the sequence of tau pathology in the inducible tau transgenic mouse model Tg4510, and in mice intracranially injected with tau-expressing AAV (adeno-associated viruses). They come up with the conclusion that caspase activation precedes and leads to neurofibrillary tangles. The data would even suggest that tangles are almost something like a salvage pathway.

The authors also conclude, based on a combination of staining methods, that tangles develop within a day’s time, which is highly reminiscent of their earlier findings, also published in Nature, that amyloid plaques develop within the same time frame (Meyer-Luehmann et al., 2008). The question is, What happens in human disease? Interestingly, in the model presented in the supplement (Supplementary Figure 6), the authors present a “Tangle formation hypothesis” that would operate in tau-overexpressing mice, but not necessarily in humans. Specifically, the authors state that “Expression of human tau (WT or P301L mutant) in a mouse brain, either by transgenesis or viral infection, leads to mislocalization of protein tau in the neuronal cell bodies.”

Hyman and colleagues, in the first part of the manuscript, count caspase-positive neurons and tangle-bearing neurons, and by determining the relative ratios (90 percent of tangle-bearing neurons are not caspase-positive versus 88 percent of caspase-positive, tangle-negative cell developing a tangle within 24 hours), they conclude that tangle-bearing neurons survive an initial caspase attack and then become caspase-negative. They also identify soluble tau rather than tangles as the culprit in AD. What is missing and what should be addressed in future studies is biochemical evidence backing these findings. Also, while images are shown representing some neurons with soluble tau and others (the end-stage) containing mature tangles (as evidenced by thioflavin S positivity), what happens in between is something like a black box. What is considered as a cell that is tangle-free could, in fact, contain already a lot of fibrillar tau, which however has not reached a level that would be detected by thioflavin S. Here, clearly, electron microscopy or Western blotting of sarkosyl or formic acid-extracted brain tissue would be helpful.

What is only mentioned in passing is that caspase-positive neurons are not observed in APP transgenic mice. It would be worthwhile determining whether there are synergistic effects in mice with a tau and Aβ pathology.

While there is an emphasis on truncation of tau, hyperphosphorylation is not considered as an initial step. It is reasonable to assume that the final word is not yet spoken. A problem, certainly, is that it is generally difficult to reduce pathological effects to (tau or Aβ) species that are underrepresented and either escape detection because of this underrepresentation (see, e.g., Aβ*56; Lesné et al., 2006) or because their defining identity is not known.

A proof whether tangle formation is preceded by caspase cleavage of soluble tau, as the paper would suggest, would be best addressed by crossing tau mutant mice onto a caspase null background.

View all comments by Jurgen Goetz

In response to concerns about the caspase inhibitor, we performed a control experiment, at the beginning of our study, to determine whether FLICA indicator inhibits caspase activation in vivo for an extended period. For this experiment, we used sequentially two different colors of FLICA.

A craniotomy was performed on an eight-month-old Tg4510 animal, and a solution of red FLICA was applied in the same conditions described in the paper. Caspase-positive cells were imaged. After one hour, a solution of green FLICA was applied. Re-imaging the same sites of the brain showed that caspase-positive cells were labeled with both colors, indicating that, within one hour of the initial application of the FLICA reagent, new caspase activity could be detected.

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 it.

View all comments by Alix de Calignon
View all comments by Bradley Hyman

There is a good chance that caspases play a physiological role in synaptic plasticity. Mattson and colleagues (1998a; 1998b) showed that activated caspases could be detected in synaptosome preparations and distal dendrites of intact cells. Although their interpretation was focused on pathology, they also concluded that "[o]ur data suggest the possibility that 'synaptic apoptosis' can occur independently of the cell body." This may mean that a caspase-dependent mechanism participates in removal of dendritic spines during normal synaptic remodeling. One of the triggers for such synaptic caspase activation is, in fact, a neurotransmitter. Thus, it should not be too surprising that neurons can persist for several hours or days after caspase activation. Whole-cell apoptosis may be something of an accidental overrun of physiological caspase activity into a quantitative state that crosses a toxic threshold.

Perhaps it should also be considered that the tau in dendrites is phosphorylated at different sites from that found in axons. This may exist to permit normal caspase activity in...  Read more

There is a good chance that caspases play a physiological role in synaptic plasticity. Mattson and colleagues (1998a; 1998b) showed that activated caspases could be detected in synaptosome preparations and distal dendrites of intact cells. Although their interpretation was focused on pathology, they also concluded that "[o]ur data suggest the possibility that 'synaptic apoptosis' can occur independently of the cell body." This may mean that a caspase-dependent mechanism participates in removal of dendritic spines during normal synaptic remodeling. One of the triggers for such synaptic caspase activation is, in fact, a neurotransmitter. Thus, it should not be too surprising that neurons can persist for several hours or days after caspase activation. Whole-cell apoptosis may be something of an accidental overrun of physiological caspase activity into a quantitative state that crosses a toxic threshold.

Perhaps it should also be considered that the tau in dendrites is phosphorylated at different sites from that found in axons. This may exist to permit normal caspase activity in dendrites from initiating toxic events stemming from tau modification. Maybe pathology arises from some unfortunate combination of caspase activation with incorrect phosphorylation patterns on tau.

References:
Mattson MP, Partin J, Begley JG. Amyloid beta-peptide induces apoptosis-related events in synapses and dendrites. Brain Res. 1998a Oct 5;807(1-2):167-76. Abstract

Mattson MP, Keller JN, Begley JG. Evidence for synaptic apoptosis. Exp Neurol. 1998b Sep;153(1):35-48. Abstract

View all comments by Steve Barger

The findings of Alix de Calignon and colleagues are interesting and support several studies using human AD tissue that show caspase plays a pivotal role in tangle formation. However, given the recent indications that AD has a preclinical phase of between 20-30 years, it remains unknown whether tangles develop within the same relatively short time frame in the human condition as suggested to occur in the brains of Tg4510 mutant human tau transgenic mice. It would have been interesting to determine whether the reported mouse tangle displays filamentous tau at the age examined, which would add additional support to the argument that the evolution of NFT pathology described in the current paper actually produces a true AD-like tangle. Despite these questions, I compliment the authors on an excellent paper!

View all comments by Elliott Mufson

Evading Apoptosis in Alzheimer Disease
Comment by Baiyang Sheng, Hyoung-gon Lee, George Perry, Mark A. Smith, Xiongwei Zhu

Using multiphoton live brain imaging in a mouse model of Alzheimer disease (AD), Hyman and colleagues were able to investigate the in vivo relationship between caspase activation and neurofibrillary tangle (NFT) formation. They suggest a new model whereby the accumulation of free cytosolic tau activates caspases; thereafter, caspase activation cleaves tau to initiate NFT formation, truncated tau recruits normal tau to misfold and form NFT. Neurons that contain NFT were unexpectedly long lived and remain alive, indicating that NFT-forming neurons escape from apoptosis.

These findings provide compelling support for a number of controversial concepts. First, the term “abortive apoptosis” (1), abbreviated as “abortosis,” was first proposed several years ago as a mechanistic explanation for the disconnect between apoptotic mediators and effectors in AD (2), as well as the mathematical improbability of apoptosis playing a key role in AD (3). A...  Read more

Evading Apoptosis in Alzheimer Disease
Comment by Baiyang Sheng, Hyoung-gon Lee, George Perry, Mark A. Smith, Xiongwei Zhu

Using multiphoton live brain imaging in a mouse model of Alzheimer disease (AD), Hyman and colleagues were able to investigate the in vivo relationship between caspase activation and neurofibrillary tangle (NFT) formation. They suggest a new model whereby the accumulation of free cytosolic tau activates caspases; thereafter, caspase activation cleaves tau to initiate NFT formation, truncated tau recruits normal tau to misfold and form NFT. Neurons that contain NFT were unexpectedly long lived and remain alive, indicating that NFT-forming neurons escape from apoptosis.

These findings provide compelling support for a number of controversial concepts. First, the term “abortive apoptosis” (1), abbreviated as “abortosis,” was first proposed several years ago as a mechanistic explanation for the disconnect between apoptotic mediators and effectors in AD (2), as well as the mathematical improbability of apoptosis playing a key role in AD (3). A second important concept is that NFTs are not obligate features of neurodegeneration and may in fact be protective (4). Supporting this, tau phosphorylation typically results as a consequence of cellular stress (5) and displays antioxidant and other protective effects (6,7) that enable NFT-containing neurons to survive for a decade (8) or, as shown here, evade apoptotic processes.

With respect to the relationship between caspase activation and NFT formation, a number of aspects merit further study. For example, how does free cytosolic tau activate caspases? The authors use caspase probes selective for caspase 6, caspase 3/7, and a pancaspase indicator. However, whether initiator caspases (caspases 8 and 9) are also activated is mechanistically important to determine. Moreover, how do neurons survive caspase activation? What is the particular role of NFT in apoptosis? In the latter, it is notable that hyperphosphorylated tau, the major protein subunit of NFTs in AD, has been shown stabilize β-catenin, suggesting a possible mechanism of NFT-bearing neurons to exit from a caspase-induced apoptotic program (9).

References:
1. Raina AK, Hochman A, Zhu X, Rottkamp CA, Nunomura A, Siedlak SL, Boux H, Castellani RJ, Perry G, Smith MA (2001) Abortive apoptosis in Alzheimer's disease. Acta Neuropathol (Berl) 101, 305-310. Abstract

2. Raina AK, Hochman A, Ickes H, Zhu X, Ogawa O, Cash AD, Shimohama S, Perry G, Smith MA (2003) Apoptotic promoters and inhibitors in Alzheimer's disease: Who wins out? Prog Neuropsychopharmacol Biol Psychiatry 27, 251-254. Abstract

3. Perry G, Nunomura A, Lucassen P, Lassmann H, Smith MA (1998) Apoptosis and Alzheimer's disease. Science 282, 1268-1269. Abstract

4. Lee HG, Perry G, Moreira PI, Garrett MR, Liu Q, Zhu X, Takeda A, Nunomura A, Smith MA (2005) Tau phosphorylation in Alzheimer's disease: pathogen or protector? Trends Mol Med 11, 164-169. Abstract

5. Su B, Wang X, Drew KL, Perry G, Smith MA, Zhu X (2008) Physiological regulation of tau phosphorylation during hibernation. J Neurochem 105, 2098-2108. Abstract

6. Rottkamp CA, Raina AK, Zhu X, Gaier E, Bush AI, Atwood CS, Chevion M, Perry G, Smith MA (2001) Redox-active iron mediates amyloid-beta toxicity. Free Radic Biol Med 30, 447-450. Abstract

7. Takeda A, Perry G, Abraham NG, Dwyer BE, Kutty RK, Laitinen JT, Petersen RB, Smith MA (2000) Overexpression of heme oxygenase in neuronal cells, the possible interaction with Tau. J Biol Chem 275, 5395-5399. Abstract

8. Morsch R, Simon W, Coleman PD (1999) Neurons may live for decades with neurofibrillary tangles. J Neuropathol Exp Neurol 58, 188-197. Abstract

9. Li HL, Wang HH, Liu SJ, Deng YQ, Zhang YJ, Tian Q, Wang XC, Chen XQ, Yang Y, Zhang JY, Wang Q, Xu H, Liao FF, Wang JZ (2007) Phosphorylation of tau antagonizes apoptosis by stabilizing beta-catenin, a mechanism involved in Alzheimer's neurodegeneration. Proc Natl Acad Sci U S A 104, 3591-3596. Abstract

View all comments by Hyoung-gon Lee
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View all comments by Xiongwei Zhu

This is a provocative study. It clearly supports the idea that caspase activation lies upstream of tangle evolution. Much of the data in this study validates what our group has been investigating in the postmortem AD brain for the past 10 years.

As far back as 2001 (Rohn et al., 2001), we put forth a hypothesis that caspase activation and cleavage of tau are early events that may precede tangle formation. We confirmed this idea in a 2002 study (Rohn et al., 2002), whereby we were the first to demonstrate the caspase-cleavage of tau in the human AD brain. In this paper, we actually provided data involving caspase-9 in the human AD brain that are now explained by Brad Hyman's group. In our 2002 study, a quantitative analysis indicated that as the number of neurons containing neurofibrillary tangles (NFTs) increased, the extent of caspase-9 activation decreased, supporting the idea that caspase-9 activation may precede NFT formation. As Hyman and colleagues show, caspase activation is initiated, but then for some reason is no longer evident, in the same tangle-bearing neuron,...  Read more

This is a provocative study. It clearly supports the idea that caspase activation lies upstream of tangle evolution. Much of the data in this study validates what our group has been investigating in the postmortem AD brain for the past 10 years.

As far back as 2001 (Rohn et al., 2001), we put forth a hypothesis that caspase activation and cleavage of tau are early events that may precede tangle formation. We confirmed this idea in a 2002 study (Rohn et al., 2002), whereby we were the first to demonstrate the caspase-cleavage of tau in the human AD brain. In this paper, we actually provided data involving caspase-9 in the human AD brain that are now explained by Brad Hyman's group. In our 2002 study, a quantitative analysis indicated that as the number of neurons containing neurofibrillary tangles (NFTs) increased, the extent of caspase-9 activation decreased, supporting the idea that caspase-9 activation may precede NFT formation. As Hyman and colleagues show, caspase activation is initiated, but then for some reason is no longer evident, in the same tangle-bearing neuron, similar to what we found with activated caspase-9 in the human AD brain.

We believe the time has come to test whether caspase inhibitors are a viable approach for the treatment of AD. Personally, it has been very difficult to obtain funding for such studies even in mouse models of AD due to concerns by reviewers regarding toxicity. But until studies are actually carried out testing whether caspase inhibitors work, one can only speculate on the relative risk versus reward of using caspase inhibitors as therapeutic agents (Rohn, 2010).

References:
Rohn TT, Head E, Su JH, Anderson AJ, Bahr BA, Cotman CW, Cribbs DH. Correlation between caspase activation and neurofibrillary tangle formation in Alzheimer's disease. Am J Pathol. 2001 Jan;158(1):189-98. Abstract

Rohn TT, Rissman RA, Davis MC, Kim YE, Cotman CW, Head E. Caspase-9 activation and caspase cleavage of tau in the Alzheimer's disease brain. Neurobiol Dis. 2002 Nov;11(2):341-54. Abstract

Rohn TT. The role of caspases in Alzheimer's disease; potential novel therapeutic opportunities. Apoptosis. 2010 Feb 3. Abstract

View all comments by Troy Rohn