The 3×Tg-AD mouse model is a sophisticated reproduction of key AD histopathological features achieved through the combined overexpression of genetically determined AβPP, PS, and tau mutations. Although producing a pathological phenotype grossly similar to that of sporadic AD, they do not replicate many aspects of this neurodegenerative disorder. This in part may be explained by the observation that mice have substantially more proteolytic enzymes, despite a genome 14% smaller than the human [25
]. Moreover, humans and mice have evolutionary diverged in regards to the age-related expression pattern of neuronal genes, being repressed in humans and rhesus macaques [27
The differences between human and Tg mice could also be due to alternative AβPP degradation pathways elicited by an overwhelming expression of mutant foreign transgenes. Familial PS and AβPP mutations produce early clinical onset, a rapid disease evolution and premature death. Presenilin mutations yield an impaired γ-secretase activity that ultimately not only affects AβPP processing, but impacts at least 25 additional substrates, among them Notch-1, N-cadherin, Erb-B4, and low density lipoprotein receptor-related protein that participate in a large number of vital cellular functions [28
]. Analysis of homogenates from 3×Tg-AD mice by WB demonstrated that the CT99 and CT83 protein bands resulting from AβPP cleavage by the β- and α-secretases respectively, which are prominent in humans, were barely detectable in this animal model. In the 3×Tg-AD mice, a novel peptide of 10 kDa was evident in the WB of the HPLC fraction 8 from the 12–16 and 18 months old mice. It is possible that this band corresponds to the 49-amino acid long AICD, resulting from γ-secretase hydrolysis at the peptide bond between amino acid residues 645-646 (ε-site) of AβPP695
. This band could also represent alternative CT-AβPP AICD peptide resulting from the γ-secretase hydrolysis that generates the Aβ40
peptides. A direct consequence of an increased synthesis and degradation of AβPP in the 3×Tg-AD mice will be a concurrent elevation of AICD peptides which may have deleterious consequences for neuronal homeostasis and promote deficits in working memory [30
Other prominent CT-AβPP fragments are represented by the 40 and 35 kDa bands which theoretically correspond to peptides of ~360 and ~320 amino acids. These longer CT-AβPP fragments are also present in the WB of human and 3×Tg-AD mouse brain homogenates, suggesting that prior to the β-secretase cleavage, other hydrolysis sites along the AβPP molecule yield extended CT fragments and potentially generate reciprocal AβPP N-terminal peptides that may serve as ligands for the death receptor-6 [31
]. In addition, if the generation of the 40 kDa peptide precedes the β- and γ-secretase cleavage, the production of this larger CT-AβPP peptide may be a potential limiting factor for the generation of Aβ peptides and hence an alternative target for therapeutic intervention.
Transgenic mice have demonstrated a strain-specific heterogeneity in the proteolytic processing of AβPP [32
]. Van Nostrand and colleagues [33
] used the C57BL/6 mouse background to create the TgSwDI mouse and, as we previously established [21
], the pattern of AβPP degradation is very similar to that exhibited by 3×Tg-AD mice. Hence, the observed differences between humans and 3×Tg-AD mice in CT-AβPP processing could reflect the use of the C57BL/6 mouse strain. Intriguingly, when using the CT9AβPP antibody in WB of the TgCRND8 mice which uses the C3 H/B6 background, also revealed lower molecular weight bands [34
] similar to those observed in the 3×Tg-AD and TgSwDI Tg mice. Mass spectrometric analysis and immunoprecipitation of these peptides in the TgCRND8 mouse using the monoclonal antibody 369, raised against the 50 CT amino acids of AβPP, suggest correspondence between the CT-AβPP peptides produced by γ-secretase hydrolysis at the γ- and ε-sites [34
]. Given the apparent differences in CT-AβPP processing between Tg mice and humans, whether some strains of AβPP/Aβ/PS Tg mice are faithful and reliable models for the testing of drugs intended to inhibit β- and γ-secretase activities in AD is questionable.
In the 3×Tg-AD mice, the noticeably decreased levels of total tau as these mice age maybe due to diminished net production of tau caused by an increased rate of degradation or decreased solubility as it is being converted to PHF. Similar results were previously observed in the 3×Tg-AD mice with IHC staining (HT7 antibody) where tau was detectable at 2–3 months, followed by a decrease starting at 12 months, and thereafter remaining constant as the mice aged [17
]. Likewise, the decrease in p-tau may be due to alterations in kinase and phosphatase activity ratios. In our experience, the amount of soluble tau recovered from enriched human NFT after extraction with either SDS or GHCl or GDFA, represents only a small fraction of the total tau present in the AD brain. This is probably due to extreme insolubility, lipid complexation and molecular cross-linkage conditions that hinder isolation. Furthermore, AD electron microscopy has revealed that PHF are apparently derived from stacks of coalescing organelles, such as degenerating mitochondria and endoplasmic reticulum [35
]. It is possible that the pathophysiological mechanisms behind the production of PHF in sporadic AD and the 3×Tg-AD mouse model are fundamentally different, since in humans, PHF result from a long standing disease process while in Tg mice, pathology results from the forced introduction of a mutant human TauP301L
gene product specifically responsible for frontotemporal dementia P-17. In humans, this tau mutation does not produce Aβ plaques and has a distinctly different clinical presentation than either sporadic or familial AD. Histological observations show that as the 3×Tg-AD mice age, they develop plaques and PHF that are detectable with the same histochemical techniques used on human tissue, and are therefore similar on a gross histochemical basis. However, the apparent similarities exhibited by the 3×Tg-AD mice require further investigation in terms of expression patterns, cleavage products, and degradation pathways to determine whether the mechanistic underpinnings of these signatures are consistent with what is known for sporadic AD.
The differences between mouse and human suggest that Tg animals reflect neither native protein product stoichiometry nor the consequential pathological conditions prevailing in sporadic AD. Although 3×Tg-AD mice, as well as other engineered AD mice, are effective models for studying mutant familial AD transgene overexpression, they cannot be expected to completely reproduce the conditions or full spectrum of pathology of sporadic AD, which is not the consequence of mutations or significantly upregulated gene expression. Furthermore, there is no reason to doubt that the mutated AβPP, PS and tau transgenes present in the 3×Tg-AD mice are in temporal terms co-expressed, a condition that may not be encountered in sporadic AD. In addition, plaques are dynamic [36
] but more permanent in humans than in Tg mice, perhaps due to the numerous post-translational modifications and complex association with glycoproteins and glycolipids [37
]. Once amyloid plaques are formed in humans they may not be remediable by any means other than by specific antibodies.
Nearly every therapeutic intervention intended to slow the progression of sporadic AD has some basis in the amyloid cascade hypothesis and extrapolates efficacy data from mouse models of familial AD directly to human sporadic AD. Heavy reliance on a single and controversial hypothesis, which is linked to a biomarker that does not correlate well with clinical dementia [41
], and the observations derived from incomplete models of human disease may be factors contributing to the disappointing outcomes of late-stage clinical trials. The field of AD has been focused on a dogmatic hypothesis that advances the presence of amyloid plaques and tangles as the ultimate target to defeat rather than inquiring about the numerous possible reasons underlying their appearance. So far, Aβ immunotherapy clinical trial data have suggested that amyloid plaques, although pathophysiologically important in AD, are not the primary dementia culprit. Furthermore, the existence of non-demented individuals with high number of pathological lesions that equate those observed in AD [41
] also detracts from the possibility of amyloid plaques as the ultimate perpetrator for the AD dementia. The ultimate conquest of AD dementia may demand meticulous scrutiny of the entire constellation of assumptions regarding fundamental aspects of dementia causality.