In this elegant paper, Rohn and colleagues show that overexpressing the anti-apoptotic protein Bcl-2 prevents the development of Alzheimer-like pathology and cognitive deficits in the 3xTg mouse model of AD. These data strengthen the growing body of evidence that caspase cleavage of tau seeds fibrillization and is necessary for NFT formation. Our ongoing studies in another mouse model of tauopathy (rTg4510) using in vivo multiphoton imaging also show an association between caspase activation and neurofibrillary tangle pathology (Spires-Jones et al., 2008).

Because the 3xTg model does not undergo neuronal loss, this paper does not contribute to the discussion of whether apoptosis is involved in cell death in AD, but the recovery of cognitive function with Bcl-2 overexpression raises the very interesting possibility that anti-apoptotic factors can improve cognition without preventing neuronal loss. Both plaques and tangles are associated with synapse loss and abnormalities in neurite architecture, so preventing the formation of...  Read more

In this elegant paper, Rohn and colleagues show that overexpressing the anti-apoptotic protein Bcl-2 prevents the development of Alzheimer-like pathology and cognitive deficits in the 3xTg mouse model of AD. These data strengthen the growing body of evidence that caspase cleavage of tau seeds fibrillization and is necessary for NFT formation. Our ongoing studies in another mouse model of tauopathy (rTg4510) using in vivo multiphoton imaging also show an association between caspase activation and neurofibrillary tangle pathology (Spires-Jones et al., 2008).

Because the 3xTg model does not undergo neuronal loss, this paper does not contribute to the discussion of whether apoptosis is involved in cell death in AD, but the recovery of cognitive function with Bcl-2 overexpression raises the very interesting possibility that anti-apoptotic factors can improve cognition without preventing neuronal loss. Both plaques and tangles are associated with synapse loss and abnormalities in neurite architecture, so preventing the formation of these pathological hallmarks may well prevent loss of connectivity that leads to cognitive decline. It will be very interesting to see data about whether synapse loss is prevented with Bcl-2 expression.

View all comments by Tara Spires

This exciting report led by Troy Rohn at Boise State University and Elizabeth Head at UC Irvine quite convincingly shows a remarkable effect of Bcl-2 overexpression in curbing Aβ and tau pathology in the brain of 3xTg-AD mice. The authors elegantly show that this action of Bcl-2 involves reducing caspase-9 activation, but is independent of prevention of neuronal death which, as in many other AD transgenic mouse models, is not present in 3xTg-AD mice to any significant degree. This is an important in vivo insight into the still poorly characterized role of Bcl-2 in cell signaling beyond its regulation of apoptosis.

In collaboration with David Morgan at the University of South Florida at Tampa, we reported that endogenous Bcl-2 is upregulated in the CNS of APP/PS1 doubly transgenic mice and that such upregulation may be neuroprotective (Karlnoski et al., 2007). Rohn and Head used an anti-Bcl-2 antibody that detects the exogenously expressed human Bcl-2 but does not recognize mouse Bcl-2. Given the lack of neuronal death in the 3xTg-AD mice, it would be interesting in the...  Read more

This exciting report led by Troy Rohn at Boise State University and Elizabeth Head at UC Irvine quite convincingly shows a remarkable effect of Bcl-2 overexpression in curbing Aβ and tau pathology in the brain of 3xTg-AD mice. The authors elegantly show that this action of Bcl-2 involves reducing caspase-9 activation, but is independent of prevention of neuronal death which, as in many other AD transgenic mouse models, is not present in 3xTg-AD mice to any significant degree. This is an important in vivo insight into the still poorly characterized role of Bcl-2 in cell signaling beyond its regulation of apoptosis.

In collaboration with David Morgan at the University of South Florida at Tampa, we reported that endogenous Bcl-2 is upregulated in the CNS of APP/PS1 doubly transgenic mice and that such upregulation may be neuroprotective (Karlnoski et al., 2007). Rohn and Head used an anti-Bcl-2 antibody that detects the exogenously expressed human Bcl-2 but does not recognize mouse Bcl-2. Given the lack of neuronal death in the 3xTg-AD mice, it would be interesting in the future to determine whether this mouse model, too, displays upregulated endogenous Bcl-2 in response to the amyloid and tau pathology. Be that as it may, Rohn and Head clearly show that the excess Bcl-2 ameliorates neuropathology and cognition in 3xTg-AD mice and propose that Bcl-2 may act by alleviating synaptic dysfunction. This is a very interesting idea that, considering the reported growing role of synaptic (dys)function in the etiology of symptomatic AD, well deserves further consideration. We have recently reported that calcineurin, a phosphatase abundant in CNS neurons and negatively involved in the modulation of synaptic plasticity and memory function, is upregulated in the CNS of singly APP transgenic mice (Tg2576) and that acute pharmacological normalization of calcineurin reverses the deficits in the fear conditioning memory task that are normally found in these mice (Dineley et al., 2007). Interestingly, in neuronal tissue, Bcl-2 has been reported to bind to CaN and sequester it (Erin et al., 2003). On this account, whether overexpressed Bcl-2 may contrast calcineurin activity in the brain of 3xTg-AD mice and so alleviate synaptic dysfunction would be a formidable question to ask.

In summary, I believe that this report may reshape our way of looking at and thinking of Bcl-2 in the context of AD-associated neuropathology. I also think that this report might have opened an interesting can of worms. And these are good worms, the kind you would like to have when going fishing for a big catch.

References:
Karlnoski R, Wilcock D, Dickey C, Ronan V, Gordon MN, Zhang W, Morgan D, Taglialatela G. Up-regulation of Bcl-2 in APP transgenic mice is associated with neuroprotection. Neurobiol Dis. 2007 Jan;25(1):179-88. Abstract

Dineley KT, Hogan D, Zhang WR, Taglialatela G. Acute inhibition of calcineurin restores associative learning and memory in Tg2576 APP transgenic mice. Neurobiol Learn Mem. 2007 Sep;88(2):217-24. Abstract

Erin N, Bronson SK, Billingsley ML. Calcium-dependent interaction of calcineurin with Bcl-2 in neuronal tissue. Neuroscience. 2003;117(3):541-55. Abstract

View all comments by Giulio Taglialatela

APP Cytoplasmic Domain: An Orphan No More!
A central tenet of the “amyloid cascade hypothesis” posits that Aβ peptides are the causative agent of AD pathogenesis. Although the details remain sketchy, the amyloid hypothesis suggests that abnormal accumulation of Aβ peptides triggers a cascade of events that cause synaptic loss and cell death, resulting ultimately in AD. Indeed, genetic data strongly implicate Aβ in AD as the FAD mutations in APP mostly flank the Aβ region and a majority of FAD mutations are found in the presenilin gene, which encodes the key component of the γ-secretase complex. Although the initial notion that the senile plaques—the accumulated stores of Aβ peptides—cause AD has now gone out of favor, a current view favors Aβ oligomers to be the real culprit. The wealth of literature implicating Aβ in AD notwithstanding, it is clear that we do not know how or which form of Aβ causes AD and whether Aβ alone can account for all facets of AD pathogenesis. This paper by Galvan et al. (1) in the...  Read more

APP Cytoplasmic Domain: An Orphan No More!
A central tenet of the “amyloid cascade hypothesis” posits that Aβ peptides are the causative agent of AD pathogenesis. Although the details remain sketchy, the amyloid hypothesis suggests that abnormal accumulation of Aβ peptides triggers a cascade of events that cause synaptic loss and cell death, resulting ultimately in AD. Indeed, genetic data strongly implicate Aβ in AD as the FAD mutations in APP mostly flank the Aβ region and a majority of FAD mutations are found in the presenilin gene, which encodes the key component of the γ-secretase complex. Although the initial notion that the senile plaques—the accumulated stores of Aβ peptides—cause AD has now gone out of favor, a current view favors Aβ oligomers to be the real culprit. The wealth of literature implicating Aβ in AD notwithstanding, it is clear that we do not know how or which form of Aβ causes AD and whether Aβ alone can account for all facets of AD pathogenesis. This paper by Galvan et al. (1) in the May 2 issue of the PNAS adds an interesting twist to this story and reminds us to keep an open mind when thinking of AD pathogenesis.

Galvan et al. present an intriguing observation that introduction of a point mutation in the cytoplasmic domain of a FAD mutant APP prevents the AD-like pathology and behavioral deficits without a reduction in Aβ levels or plaque deposition. Dale Bredesen, the senior author of the study, and Eddie Koo had previously reported (2) that the consensus caspase cleavage site (661VEVD664, numbering according to APP695) in the APP cytoplasmic domain may be cleaved when the apoptotic program is activated (also independently shown by Gervais et al. [3]). Koo and Bredesen also demonstrated that C-terminal 31-residue-long peptide comprising APP 665-695 (termed C31), a product of such a cleavage event, was proapoptotic and put forward a model (4) based on cell culture data in which Aβ binds and dimerizes APP, triggering the cleavage of the APP at the VEVD motif and releasing the cytotoxic C31 fragment. Interestingly, the work of Bernadette Allinquant (5) suggests that the upstream peptide comprising APP 649-664 (termed Jcasp), also a product of cleavage at D664, activates caspases and induces neuronal death.

To determine the contribution of this cleavage event to AD pathogenesis, the Bredesen group made use of the PDAPP mouse model of AD. These mice express human APP bearing the Swedish and Indiana mutations, produce higher amounts of Aβ40 and Aβ42, and develop Aβ plaques by 12-15 months of age. These mice also show synaptic loss, astrogliosis, dentate gyral atrophy, and behavioral deficits in Morris water maze and Y-maze tests. Galvan et al., introduced a mutation changing Asp664 to Ala in the APP cytoplasmic domain, which is presumably resistant to cleavage by caspases. The authors report that their “triple”-transgenic mice (bearing the Swedish, Indiana, and D664A mutations) produced comparable amounts of Aβ and formed similar plaque load when compared to PDAPP mice. Unexpectedly, introduction of the D664A mutation completely prevented synaptic loss, astrogliosis, and memory deficits seen in PDAPP mice. The authors conclude that the APP cytoplasmic domain plays a key role in the development of AD-like deficits in transgenic mice. The authors claim that this observation represents a prevention or reversal of AD-like pathology and behavioral deficits. However, keeping in mind that these are independently generated lines with different integration sites, it is perhaps more appropriate to conclude that these mice fail to show the expected deficits. It should be noted that the authors do present data from two independent lines, and both show a lack of behavioral deficits.

Since we do not know how D664A mutation counters the AD-like deficits in PDAPP mice, the findings of Galvan et al. can be interpreted in many ways. Some might conclude these data to show that Aβ plays no or only a minor role in the development of AD pathogenesis. However, others may point to the wealth of literature implicating Aβ in AD and dismiss such a drastic conclusion. The authors seem to favor the view that the cleavage of the APP cytoplasmic domain at D664 lies downstream from events initiated by Aβ. Another possibility is that D664A mutation alters the binding of the APP cytoplasmic domain with its interacting partners and thereby prevents the deleterious effects observed in PDAPP mice. One issue the authors did not consider is what happens to APP intracellular domain (AICD) produced from D664A bearing APP in their transgenic mice. There is increasing awareness that AICD could play a significant role in APP function and/or AD pathogenesis (6). Since D664A mutation results in the loss of a negative charge and takes place near the T668 residue (phosphorylation of which alters binding with Fe65), it is not unlikely that D664A mutation results in major alterations in protein-protein interaction involving both the APP cytoplasmic domain and AICD.

The effects of D664A mutation in preventing AD-like deficits are so striking that these studies are likely to be repeated in other mouse models of AD. Although the cellular basis of the effects of D664A mutation in blocking the synaptic loss and behavioral deficits remains obscure, these findings demonstrate a key role for APP cytoplasmic domain (and/or AICD) in AD pathogenesis in the PDAPP mouse model. Irrespective of whether it plays a role downstream of Aβ or independent of Aβ, these findings further add to the increasing importance of APP cytoplasmic domain/AICD in APP pathophysiology. This paper also shows that a more open view of AD pathogenesis will be a welcome change.

References:
1. Galvan V, Gorostiza OF, Banwait S, Ataie M, Logvinova AV, Sitaraman S, Carlson E, Sagi SA, Chevallier N, Jin K, Greenberg DA, Bredesen DE. Reversal of Alzheimer's-like pathology and behavior in human APP transgenic mice by mutation of Asp664. Proc Natl Acad Sci U S A. 2006 May 2;103(18):7130-5. Epub 2006 Apr 25. Abstract

2. Lu DC, Rabizadeh S, Chandra S, Shayya RF, Ellerby LM, Ye X, Salvesen GS, Koo EH, Bredesen DE. A second cytotoxic proteolytic peptide derived from amyloid beta-protein precursor. Nat Med. 2000 Apr;6(4):397-404. Abstract

3. Gervais FG, Xu D, Robertson GS, Vaillancourt JP, Zhu Y, Huang J, LeBlanc A, Smith D, Rigby M, Shearman MS, Clarke EE, Zheng H, Van Der Ploeg LH, Ruffolo SC, Thornberry NA, Xanthoudakis S, Zamboni RJ, Roy S, Nicholson DW. Involvement of caspases in proteolytic cleavage of Alzheimer's amyloid-beta precursor protein and amyloidogenic A beta peptide formation. Cell. 1999 Apr 30;97(3):395-406. Abstract

4. Lu DC, Shaked GM, Masliah E, Bredesen DE, Koo EH. Amyloid beta protein toxicity mediated by the formation of amyloid-beta protein precursor complexes. Ann Neurol. 2003 Dec;54(6):781-9. Abstract

5. Madeira A, Pommet JM, Prochiantz A, Allinquant B. SET protein (TAF1beta, I2PP2A) is involved in neuronal apoptosis induced by an amyloid precursor protein cytoplasmic subdomain. FASEB J. 2005 Nov;19(13):1905-7. Epub 2005 Sep 14. Abstract

6. Ryan KA, Pimplikar SW. Activation of GSK-3 and phosphorylation of CRMP2 in transgenic mice expressing APP intracellular domain. J Cell Biol. 2005 Oct 24;171(2):327-35. Epub 2005 Oct 17. Abstract

View all comments by Sanjay W. Pimplikar

I have read this article with great interest since I believe it is always refreshing to hear about alternative views that explain the deficits characteristic of Alzheimer disease. This paper provides evidence that, whereas amyloid production and plaque formation were unaltered, synaptic loss and behavioral abnormalities were completely prevented by mutation at a functional Asp664 caspase cleavage site. This site was described in a previously published paper from Konrad Beyreuther’s laboratory in Heidelberg (see Weidemann et al., 1999) and was suggested to regulate programmed cell death. Meanwhile, we know that this site is within the APP intracellular domain, which has been named AICD and which consists of the last 50 carboxy-terminal residues of the APP protein.

The AICD fragment, like NICD, can complex with transcription factors. Unfortunately, since the authors did not pay attention to the AICD molecule, we do not know what effect the D664A mutation has on the AICD production, stability and its transport to the nucleus. While...  Read more

I have read this article with great interest since I believe it is always refreshing to hear about alternative views that explain the deficits characteristic of Alzheimer disease. This paper provides evidence that, whereas amyloid production and plaque formation were unaltered, synaptic loss and behavioral abnormalities were completely prevented by mutation at a functional Asp664 caspase cleavage site. This site was described in a previously published paper from Konrad Beyreuther’s laboratory in Heidelberg (see Weidemann et al., 1999) and was suggested to regulate programmed cell death. Meanwhile, we know that this site is within the APP intracellular domain, which has been named AICD and which consists of the last 50 carboxy-terminal residues of the APP protein.

The AICD fragment, like NICD, can complex with transcription factors. Unfortunately, since the authors did not pay attention to the AICD molecule, we do not know what effect the D664A mutation has on the AICD production, stability and its transport to the nucleus. While the authors claim in their paper that Asp664 does not affect Aβ production but is critical for synaptic loss, this conclusion is based on analysis of soluble Aβ by ELISA that detects Aβ40 and Aβ42, and by Western analysis. Here a biochemist would like to see a more detailed analysis by MS, etc. It would also be of interest to see how intracellular Aβ is affected since intraneuronal Aβ accumulation precedes plaque formation in APP and PS1 double-transgenic mice (see Wirths et al., 2001). At the end, it may turn out that the authors are right to believe that the cleavage of APP at Asp664 represents a therapeutic target but possibly is also due to effects on the consecutive cleavage mechanism of γ-secretase (see Qi-Takahara et al., 2005) or intracellular Aβ species, or both.

View all comments by Gerd Multhaup

Amyloid-β: The Finger or the Moon?
The study by Galvan et al. (Galvan et al., 2006) provides another clear example that amyloid-β is not responsible for the cognitive and pathological changes that stereotypify Alzheimer disease (AD) (Lee et al., 2004; Lee et al., 2006). Specifically, the researchers demonstrate that by introducing an additional mutation to PDAPP mice that prevents the cleavage of APP by caspase, while not affecting amyloid-β, rescues cognitive and pathological deficits in PDAPP transgenic mice. Taken together with recent findings that knockout of presenilin 1 (i.e., no amyloid-β), while abrogating amyloid-β pathology from APP mutant transgenic mice, failed to rescue cognitive deficits (Dewachter et al., 2002; Saura et al., 2005), there can only be one conclusion: Amyloid is not responsible for cognitive deficits. Indeed, this conclusion is based on both negative and positive correlates (i.e., cognitive deficits with no amyloid-β and rescue of cognitive deficits without change of amyloid-β). It is...  Read more

Amyloid-β: The Finger or the Moon?
The study by Galvan et al. (Galvan et al., 2006) provides another clear example that amyloid-β is not responsible for the cognitive and pathological changes that stereotypify Alzheimer disease (AD) (Lee et al., 2004; Lee et al., 2006). Specifically, the researchers demonstrate that by introducing an additional mutation to PDAPP mice that prevents the cleavage of APP by caspase, while not affecting amyloid-β, rescues cognitive and pathological deficits in PDAPP transgenic mice. Taken together with recent findings that knockout of presenilin 1 (i.e., no amyloid-β), while abrogating amyloid-β pathology from APP mutant transgenic mice, failed to rescue cognitive deficits (Dewachter et al., 2002; Saura et al., 2005), there can only be one conclusion: Amyloid is not responsible for cognitive deficits. Indeed, this conclusion is based on both negative and positive correlates (i.e., cognitive deficits with no amyloid-β and rescue of cognitive deficits without change of amyloid-β). It is surprising that the field still remains convinced that amyloid is important.

“When a sage points to the moon, some people see the finger and not the moon.”

References:
Dewachter I, Reverse D, Caluwaerts N, Ris L, Kuiperi C, Van den Haute C, Spittaels K, Umans L, Serneels L, Thiry E, Moechars D, Mercken M, Godaux E, Van Leuven F. Neuronal deficiency of presenilin 1 inhibits amyloid plaque formation and corrects hippocampal long-term potentiation but not a cognitive defect of amyloid precursor protein [V717I] transgenic mice. J Neurosci. 2002 May 1;22(9):3445-53. Abstract

Galvan V, Gorostiza OF, Banwait S, Ataie M, Logvinova AV, Sitaraman S, Carlson E, Sagi SA, Chevallier N, Jin K, Greenberg DA, Bredesen DE. Reversal of Alzheimer's-like pathology and behavior in human APP transgenic mice by mutation of Asp664. Proc Natl Acad Sci U S A. 2006 May 2;103(18):7130-5. Epub 2006 Apr 25. Abstract

Lee HG, Casadesus G, Zhu X, Takeda A, Perry G, Smith MA. Challenging the amyloid cascade hypothesis: senile plaques and amyloid-beta as protective adaptations to Alzheimer disease. Ann N Y Acad Sci. 2004 Jun;1019:1-4. Review. Abstract

Lee HG, Zhu X, Nunomura A, Perry G, Smith MA. Amyloid beta: the alternate hypothesis. Curr Alzheimer Res. 2006 Feb;3(1):75-80. Review. Abstract

Saura CA, Chen G, Malkani S, Choi SY, Takahashi RH, Zhang D, Gouras GK, Kirkwood A, Morris RG, Shen J. Conditional inactivation of presenilin 1 prevents amyloid accumulation and temporarily rescues contextual and spatial working memory impairments in amyloid precursor protein transgenic mice. J Neurosci. 2005 Jul 20;25(29):6755-64. Abstract

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