Lauritzen et al. report an excellent study that further refines our understanding of the intracellular pathology observed in the 3xTg-AD mouse model. This mouse model is unique in that immunohistochemical methods show robust accumulation of intracellular APP-related material. What is this intraneuronal material? While originally described to be intraneuronal Aβ, we (1) and others found that much of it shared epitopes that span the APP molecule, and that the presence of this material is not altered upon genetic ablation of BACE. The current paper now shows that, in addition to diffuse intracellular accumulation of full-length APP, there is an age- and region-dependent accumulation of APP C-terminal fragments in this model. The authors draw on a variety of methods including immunohistochemistry, biochemistry, enzymatic analysis, genetic manipulation, and pharmacology to support their conclusion that hippocampal neurons accumulate C99 within a lysosomal compartment. The cross-validation using multiple, distinct experimental methods adds rigor to this thorough and compelling...  Read more

Lauritzen et al. report an excellent study that further refines our understanding of the intracellular pathology observed in the 3xTg-AD mouse model. This mouse model is unique in that immunohistochemical methods show robust accumulation of intracellular APP-related material. What is this intraneuronal material? While originally described to be intraneuronal Aβ, we (1) and others found that much of it shared epitopes that span the APP molecule, and that the presence of this material is not altered upon genetic ablation of BACE. The current paper now shows that, in addition to diffuse intracellular accumulation of full-length APP, there is an age- and region-dependent accumulation of APP C-terminal fragments in this model. The authors draw on a variety of methods including immunohistochemistry, biochemistry, enzymatic analysis, genetic manipulation, and pharmacology to support their conclusion that hippocampal neurons accumulate C99 within a lysosomal compartment. The cross-validation using multiple, distinct experimental methods adds rigor to this thorough and compelling study.

This work is entirely consistent with our findings in that they observe diffuse perikaryal accumulation of material which contains epitopes that span APP, and that this intraneuronal material is not the Aβ peptide. This accumulation of intracellular APP is unique to the 3xTg mouse model; transgenic mice with even higher levels of APP expression do not show similar intracellular immunoreactivity. In addition to this diffuse intracellular APP, the authors now demonstrate that hippocampal neurons show an age-dependent accumulation of punctate immunoreactivity which appears to be predominantly C99. The current study relied in part on the polyclonal FCA18 antibody, which is reported to be specific to the N-terminus of Aβ. Most, if not all, end-specific antibodies show at best increased affinity to cleaved/free epitopes over the same sequence in the uncleaved protein. Knowing this relative affinity is critical to evaluating these results. That being said, supporting biochemical and pharmacologic evidence is also presented. Notably, we did not observe an age-dependent increase in intracellular APP accumulation. In contrast with the current study, we performed biochemical analysis only on cerebral cortical lysates in which we did not detect accumulation of C99. Lauritzen and colleagues analyzed hippocampal, cortical, and whole hemi-brain lysates, which indicate that the accumulation of C99 is region specific (i.e., occurs mainly in the hippocampus).

This accumulation is not due to increased β-secretase activity, and it can be augmented by inhibition of γ-secretase. Although the mechanisms responsible for the accumulation are unclear, these results remind me of the work of Charles Glabe and colleagues showing that uptake of extracellular Aβ can lead to lysosomal accumulation of APP fragments which were subject to non-secretase-dependent trimming (2-4). Indeed, the current study shows that the punctate immunoreactivity is not labeled with an antibody that recognizes the C-terminus of APP, suggesting that this material may be C-terminally truncated. Of course, caution is always warranted in overanalyzing negative immunohistochemistry results, as epitope masking can create false negative results.

APP CTF analysis of rodent brain is complicated by the fact that there are at least five APP CTF bands representing various phosphorylated and non-phosphorylated CTFs (5,6). The intracellular APP CTF described by Glabe and colleagues in cultured cells migrates more slowly than C99 on tris-tricine gels; C99 is typically smaller than 14.2 kDa (3). Likewise, the APP fragment identified in the current study seems to be a little larger (more than 14.2 kDa) than what is typically observed by others for C99 (less than 14.2 kDa) in cells or brain tissue (3,5). Accurate identification might come from sequencing the band, aligning it with known APP CTFs, or by using various antibodies to different epitopes to analyze it. Dephosphorylation of APP CTFs would also simplify analysis.

Finally, the question that remains is, What is the relationship between this intracellular pathology and the pathogenesis of AD? It is not clear if tau is involved. We observed tau pathology in these mice as early as five to seven months of age, even in the absence of BACE activity when no C99 is produced. Ultimately, we need better mechanistic understanding about why this mouse model demonstrates these intracellular changes. Then we can address the question of whether intraneuronal accumulation of APP, APP fragments, and/or Aβ is relevant in terms of the pathogenesis of Alzheimer’s disease. So far, experimental and human studies have been equivocal on this debate, with evidence on both sides of the fence regarding the importance of intracellular APP/APP fragment/Aβ pathology.

References:
1. Winton MJ, Lee EB, Sun E, Wong MM, Leight S, Zhang B, Trojanowski JQ, Lee VM. Intraneuronal APP, not free Aβ peptides in 3xTg-AD mice: implications for tau versus Aβ-mediated Alzheimer neurodegeneration. J Neurosci. 2011 May 25;31(21):7691-9. Abstract

2. Knauer MF, Soreghan B, Burdick D, Kosmoski J, Glabe CG. Intracellular accumulation and resistance to degradation of the Alzheimer amyloid A4/β protein. Proc Natl Acad Sci U S A. 1992 Aug 15;89(16):7437-41. Abstract

3. Yang AJ, Knauer M, Burdick DA, Glabe C. Intracellular A β 1-42 aggregates stimulate the accumulation of stable, insoluble amyloidogenic fragments of the amyloid precursor protein in transfected cells. J Biol Chem. 1995 Jun 16;270(24):14786-92. Abstract

4. Glabe C. Intracellular mechanisms of amyloid accumulation and pathogenesis in Alzheimer's disease. J Mol Neurosci. 2001 Oct;17(2):137-45. Abstract

5. Buxbaum JD, Thinakaran G, Koliatsos V, O'Callahan J, Slunt HH, Price DL, Sisodia SS. Alzheimer amyloid protein precursor in the rat hippocampus: transport and processing through the perforant path. J Neurosci. 1998 Dec 1;18(23):9629-37. Abstract

6. Lee EB, Zhang B, Liu K, Greenbaum EA, Doms RW, Trojanowski JQ, Lee VM. BACE overexpression alters the subcellular processing of APP and inhibits Aβ deposition in vivo. J Cell Biol. 2005 Jan 17;168(2):291-302. Abstract

View all comments by Edward B. Lee

This study from the Checler group provides compelling evidence that 3xTg mice accumulate the C99 fragment of APP and not Aβ at a young age (three to six months). It is likely that these results may not bring an end to the ongoing debate regarding the presence of intraneuronal Aβ (see ARF Live Discussion). However, if one accepts their interpretation, then the inescapable conclusion of this study is that it is the accumulation of C99 and not that of Aβ that correlates with the appearance of LTP impairment and memory deficits observed in young 3xTg mice (1). Such a conclusion will be consistent with the recent studies from the Nixon group (2), and the old observations of Rachel Neve (and others) that transgenic mice overexpressing C99 fragment develop AD-like pathologies (3). The recent discovery of the "protective" APP mutation (4) will also be consistent with the idea that C99 by itself is toxic and brings about AD-like pathology.

However, this study begs the question, What is the mechanism by which...  Read more

This study from the Checler group provides compelling evidence that 3xTg mice accumulate the C99 fragment of APP and not Aβ at a young age (three to six months). It is likely that these results may not bring an end to the ongoing debate regarding the presence of intraneuronal Aβ (see ARF Live Discussion). However, if one accepts their interpretation, then the inescapable conclusion of this study is that it is the accumulation of C99 and not that of Aβ that correlates with the appearance of LTP impairment and memory deficits observed in young 3xTg mice (1). Such a conclusion will be consistent with the recent studies from the Nixon group (2), and the old observations of Rachel Neve (and others) that transgenic mice overexpressing C99 fragment develop AD-like pathologies (3). The recent discovery of the "protective" APP mutation (4) will also be consistent with the idea that C99 by itself is toxic and brings about AD-like pathology.

However, this study begs the question, What is the mechanism by which C99 contributes to AD, if not as a precursor of Aβ or AICD (5)? Delineating the downstream molecular mechanisms—not an easy or quick task—will go a long way not only towards convincing the field of the importance of non-amyloidogenic mechanisms of AD (6), but also in expanding the repertoire of therapeutic strategies to treat the disease. There is no controversy about the need of the latter.

References:
1. Oddo S, Caccamo A, Shepherd JD, Murphy MP, Golde TE, Kayed R, Metherate R, Mattson MP, Akbari Y, Laferla FM. Triple-transgenic model of Alzheimer's disease with plaques and tangles: intracellular Aβ and synaptic dysfunction. Neuron. 2003 Jul 31;39(3):409-21. Abstract

2. Jiang Y, Mullaney KA, Peterhoff CM, Che S, Schmidt SD, Boyer-Boiteau A, Ginsberg SD, Cataldo AM, Mathews PM, Nixon RA. Alzheimer's-related endosome dysfunction in Down syndrome is Aβ-independent but requires APP and is reversed by BACE-1 inhibition. Proc Natl Acad Sci U S A. 2010 Jan 26;107(4):1630-5. Abstract

3. Oster-Granite ML, McPhie DL, Greenan J, Neve RL. Age-dependent neuronal and synaptic degeneration in mice transgenic for the C terminus of the amyloid precursor protein. J Neurosci. 1996 Nov 1;16(21):6732-41. Abstract

4. Jonsson T, Atwal JK, Steinberg S, Snædal J, Jonsson PV, Bjornsson S, Stefansson H, Sulem P, Gudbjartsson D, Maloney J, Hoyte K, Gustafson A, Liu Y, Lu Y, Bhangale T, Graham RR, Huttenlocher J, Bjornsdottir G, Andreassen OA, Jönsson EG, Palotie A, Behrens TW, Magnusson OT, Kong A, Thorsteinsdottir U, Watts RJ, Stefansson K. A mutation in APP protects against Alzheimer's disease and age-related cognitive decline. Nature. 2012 Aug 2;488(7409):96-9. Abstract

5. Ghosal K, Vogt DL, Liang M, Shen Y, Lamb BT, Pimplikar SW. Alzheimer's disease-like pathological features in transgenic mice expressing the APP intracellular domain. Proc Natl Acad Sci U S A. 2009 Oct 27;106(43):18367-72. Abstract

6. Pimplikar SW, Nixon RA, Robakis NK, Shen J, Tsai LH. Amyloid-independent mechanisms in Alzheimer's disease pathogenesis. J Neurosci. 2010 Nov 10;30(45):14946-54. Abstract

View all comments by Sanjay W. Pimplikar

Lauritzen et al. provide important new insights into age-related changes in APP fragments within neurons of the triple transgenic mouse. They show interesting early and age-related elevations in APP C-terminal fragments (CTFs), but not full-length APP, in these mice beginning at three months. Remarkably, they see similar elevations in CTFs in 2xTg-AD mice that lack the FAD mutant PS1 knock-in. This paper also provides further evidence for intraneuronal Aβ42 accumulation in 3xTg-AD mice that begins at six months of age, although levels appear low. They also show that with γ-secretase inhibition, brain APP CTFs increase, while β amyloid disappears.

It seems that this group now has in its hands the models to test the relative roles of APP CTFs and β amyloid, since they can compare behavior and synapse alterations in 3xTg-AD versus 2xTg-AD mice. Since CTF increases are similar in the two strains, then synaptic pathology and behavior should be similar if CTFs are the key pathogenic determinants. Alternatively, if β amyloid is more pathogenic, synapses and behavior should be less...  Read more

Lauritzen et al. provide important new insights into age-related changes in APP fragments within neurons of the triple transgenic mouse. They show interesting early and age-related elevations in APP C-terminal fragments (CTFs), but not full-length APP, in these mice beginning at three months. Remarkably, they see similar elevations in CTFs in 2xTg-AD mice that lack the FAD mutant PS1 knock-in. This paper also provides further evidence for intraneuronal Aβ42 accumulation in 3xTg-AD mice that begins at six months of age, although levels appear low. They also show that with γ-secretase inhibition, brain APP CTFs increase, while β amyloid disappears.

It seems that this group now has in its hands the models to test the relative roles of APP CTFs and β amyloid, since they can compare behavior and synapse alterations in 3xTg-AD versus 2xTg-AD mice. Since CTF increases are similar in the two strains, then synaptic pathology and behavior should be similar if CTFs are the key pathogenic determinants. Alternatively, if β amyloid is more pathogenic, synapses and behavior should be less impaired in the 2x-AD mice.

Although this work is carefully done, the question remains whether there is any accumulation of intracellular β amyloid prior to six months in 3xTg-AD mice. Lauritzen and colleagues show that γ-secretase inhibition raises brain C99 levels at three months of age. As the authors note, this means that C99 is normally processed and β amyloid is generated at this early age. Since β amyloid is generated inside cells, there should therefore be some intraneuronal β amyloid. The challenge is how one can detect it. For this we need to consider higher sensitivity/resolution methods, such as more sensitive ELISAs (e.g., Hashimoto et al., 2010) or, as we have done, immunoelectron microscopy and/or higher-resolution immunofluorescence microscopy.

References:
Hashimoto M, Bogdanovic N, Volkmann I, Aoki M, Winblad B, Tjernberg LO. Analysis of microdissected human neurons by a sensitive ELISA reveals a correlation between elevated intracellular concentrations of Aβ42 and Alzheimer's disease neuropathology. Acta Neuropathol. 2010 May;119(5):543-54. Abstract

View all comments by Gunnar K. Gouras