. Longitudinal, quantitative assessment of amyloid, neuroinflammation, and anti-amyloid treatment in a living mouse model of Alzheimer's disease enabled by positron emission tomography. J Neurosci. 2007 Oct 10;27(41):10957-68. PubMed.

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  1. Most scientists have had the experience of reading a manuscript and thinking, “Wow! I wish I had written that.” The paper by Maeda et al. contains three separate studies, all of which generated that response in me. It is, in my mind, the most impressive study of amyloid imaging agents in animals yet published.

    The first part of the study by Maeda et al. involves the vexing transgenic (Tg) mouse problem reported by our lab (Klunk et al., 2005) and that of Toyama et al. (Toyama et al., 2005). The problem lies in the fact that although the amyloid imaging agent, Pittsburgh Compound-B (PiB), shows high signal-to-noise in human brain areas known to contain high loads of amyloid-β (Aβ) plaques, almost no such signal could be detected in microPET studies using transgenic (Tg) mouse models of amyloid deposition at an age when the Aβ plaque load in these mice is several-fold higher than anything seen in human brain. We had suggested that this was based on the fact that, like synthetic Aβ aggregated in vitro, Tg mouse brain contained 1/500th the number of PiB binding sites per mole of Aβ compared to homogenates of human AD brain (Klunk et al., 2005). That is, there is something physically different about Aβ fibrils aggregated in the test tube (over days) or in Tg mouse brain (over months) compared to that aggregated in human brain (over a decade). We found that the small number of PiB binding sites in Tg mouse brain did not yield a sufficient signal-to-noise level to allow detection using the typical [11C]PiB preparations we employ for animal studies (specific activity of ~20-30 GBq/μmol).

    Maeda et al. succeeded in reliably detecting a microPET PiB signal in 20-22-month-old APP23 mice, by taking great pains to prepare and employ [11C]PiB with 10-fold higher specific activity (~300 GBq/μmol). They also showed the expected age-related increase in PiB retention from 17 to 29 months in these mice. In retrospect, this makes perfect sense, and a quick check of predicted binding signal as a function of tracer-specific activity calculated with standard receptor binding equations shows that increasing the specific activity of PiB from 30 to 300 GBq/μmol would result in the signal-to-noise ratio in Tg mouse brain microPET studies going from basically undetectable (~1.0-1.1) up to the ~1.5 range reported by Maeda and colleagues.

    The natural question, then, becomes, if we can improve amyloid imaging in mice by increasing the specific activity of [11C]PiB, can we obtain similar gains in human PiB PET? The answer to this—using the same equations mentioned above—is “no” if there is significant amyloid present. That is, with the amount of amyloid we currently detect in AD patients, “amyloid-positive” MCI patients and even “amyloid-positive” controls, [11C]PiB binding is already maximal at specific activities lower than 30 GBq/μmol. Further increases in specific activity would not be predicted to show significant increases in the signal-to-noise ratio.

    However, what about in those brains in which the amyloid load is at or just below current detection limits? In these cases, increasing the specific activity could increase the sensitivity and decrease the detection threshold for amyloid. This has several implications. It is difficult to routinely produce [11C]PiB with a specific activity of 300 GBq/μmol for human studies. In contrast, fluorine-18-labeled tracers can be produced at this level of specific activity more easily in most cases. Thus, in addition to making amyloid imaging more widely available, the development of an F-18 analogue of PiB may carry additional importance. One such F-18 PiB analogue has already shown promise in preliminary human studies (Mathis et al., 2007). The present observation by Maeda et al. could prove extremely important, and could have stood alone to make an excellent contribution to the literature.

    The second part of the study by Maeda et al. is equally able to stand alone as an important contribution to the field. In this series of experiments, Maeda et al. show that their PiB microPET protocol can be used to follow the removal of amyloid in vivo over the course of 2 weeks after intrahippocampal injection of an anti-Aβ antibody. In addition, they use the activated microglial marker [18F]FE-DAA1106 to show a corresponding increase in glial markers/neuroinflammation around the injection site. They show that this microglial response is dependent on the presence of amyloid since the injection of the anti-Aβ antibody into wild-type mice did not result in increased [18F]FE-DAA1106 retention. This series of experiments certainly has implications for the monitoring of human immunotherapy trials and actually mirrors protocols being contemplated by several pharmaceutical companies.

    The third set of experiments in the Maeda et al. paper addresses the closely related issues of why PiB binds so poorly to Tg mouse brain and what is the molecular substrate of PiB binding in human brain. The authors present evidence to suggest that C-terminal heterogeneity is not a particularly important determinant of PiB binding. PiB binds to both Aβ40 and Aβ42 plaques and probably to plaques with other C-termini as well. In exploring the contribution of N-terminal heterogeneity to PiB binding, they discovered that the levels of AβN3-pyroglutamate or “N3(pE)” were much more closely tied to PiB binding than were levels of Aβ isoforms starting at the aspartate at position-1 or “N1D.” In vitro experiments using aggregated, synthetic AβN1D-42 and AβN3(pE)-42 showed that the N3(pE) form bound PiB ~fivefold better than the N1D form. However, this experiment cannot fully explain the contribution of AβN3(pE) because this form of synthetic fibril still has less than 1/100th the number of PiB binding sites per mole of Aβ than that observed per mole of Aβ in human brain (Klunk et al., 2005). This is likely due to the artificial aggregation conditions affecting the final fibril structure. Despite this, AβN3(pE) is still an attractive candidate for an important PiB binding substrate in vivo.

    Maeda et al. point out that AβN3(pE) isoforms are abundant in AD brain. Previous reports have suggested AβN3(pE)42 may account for >25 percent of the total Aβ in human plaques and appears to aggregate more readily and produce greater toxicity than N1D forms of Aβ (Saido et al., 1995; Harigaya et al., 2000; Russo et al., 2002; Guntert et al., 2006). This finding may explain a large part of the low PiB binding in Tg mouse brain, because Tg mouse brain contains very little AβN3(pE) (Guntert et al., 2006). Saido et al. have previously stressed the importance of the AβN3(pE) isoform by noting that plaques containing AβN3(pE) are “present in equivalent or greater densities than those composed of standard Aβ…” and “because deposition of the former species [i.e., AβN3(pE)] appears to precede deposition of the latter [i.e., AβN1D], as confirmed with specimens from Down syndrome patients, the processes involved in AβN3(pE) production and retention may play an early and critical role in senile plaque formation” (Saido et al., 1995). Thus, the N3(pE) form of Aβ may play a significant role in the pathogenesis of AD and be a prime binding site for PiB. This preliminary finding will require further study before its full significance can be determined, but if true, this could imply that PiB has some specificity for an Aβ isoform that has special pathological relevance.

    In summary, the paper by Maeda et al. represents a very important set of three contributions to the literature of AD and amyloid imaging:

    1. this paper shows that amyloid deposition in Tg mice can be detected in vivo by using high specific activity PiB, and this may have implications for decreasing the detection threshold in humans;

    2. this paper shows amyloid imaging can detect the therapeutic effect of anti-amyloid immunotherapy and the reciprocal nature of neuroinflammation associated with amyloid removal; and

    3. the N3(pE) isoform of Aβ may have special pathological significance; relative differences in the abundance of AβN3(pE) in human and Tg mouse brain may explain some of the poor PiB binding in Tg mice; and N3(pE) may be a specific target of PiB in vivo.

    References:

    . High sensitivity analysis of amyloid-beta peptide composition in amyloid deposits from human and PS2APP mouse brain. Neuroscience. 2006 Dec 1;143(2):461-75. PubMed.

    . Amyloid beta protein starting pyroglutamate at position 3 is a major component of the amyloid deposits in the Alzheimer's disease brain. Biochem Biophys Res Commun. 2000 Sep 24;276(2):422-7. PubMed.

    . Binding of the positron emission tomography tracer Pittsburgh compound-B reflects the amount of amyloid-beta in Alzheimer's disease brain but not in transgenic mouse brain. J Neurosci. 2005 Nov 16;25(46):10598-606. PubMed.

    . Comparison of the amyloid imaging agents [F-18]3’-F-PIB and [C-11]PIB in Alzheimer's disease and control subjects. J Nucl Med 2007; 48:56P.

    . Pyroglutamate-modified amyloid beta-peptides--AbetaN3(pE)--strongly affect cultured neuron and astrocyte survival. J Neurochem. 2002 Sep;82(6):1480-9. PubMed.

    . Dominant and differential deposition of distinct beta-amyloid peptide species, A beta N3(pE), in senile plaques. Neuron. 1995 Feb;14(2):457-66. PubMed.

    . PET imaging of brain with the beta-amyloid probe, [11C]6-OH-BTA-1, in a transgenic mouse model of Alzheimer's disease. Eur J Nucl Med Mol Imaging. 2005 May;32(5):593-600. PubMed.

  2. Besides the interesting issues already discussed at length and in depth, the data bring to mind the problem of why N-terminally directed antibodies—and particularly those against the EFRH epitope as demonstrated by Beka Solomon and coworkers—are most efficient in passive vaccination. The explanation is that the N-terminal is "dangling" outside the amyloid fibers and thereby accessible.

    Then I wonder about antibodies that react about two orders of magnitude less well with pE-Aβ (i.e., Aβ3-42 peptide, starting with pyroglutamyl at residue Glu-3), than with wt-Aβ (Gardberg et al., 2007). Are these acting not or less well on pE-Aβ in human brain and thereby explaining differences in efficacy of passive vaccination in mouse models and human patients?

    References:

    . Molecular basis for passive immunotherapy of Alzheimer's disease. Proc Natl Acad Sci U S A. 2007 Oct 2;104(40):15659-64. PubMed.

    View all comments by Fred Van Leuven
  3. This report provides first evidence for a direct correlation of PiB (Pittsburgh Compound-B) retention analyzed by PET imaging in living APP transgenic mice. This is a very important paper, because it describes the temporal and spatial distribution of plaque deposition after intravenous injection of PiB, a compound applied in Alzheimer disease (AD) patients. The authors were able to show that passive immunization against human Aβ peptide reduced PiB retention, correlating well with an increase in glia radiotracer signaling. It is at present, however, unclear, whether the observed increase in gliosis is directly involved in Aβ phagocytosis and clearance. In any case, the passive immunization clearly shows that it has an effect on PiB retention and amyloidosis in vivo. Of special interest, the PiB binding best correlated with plaques positive for N-terminally truncated and modified Aβ, Aβ-N3-pyroglutamate (AβN3[pE]) in AD brain and three different APP transgenic mouse models.

    The existence of N-terminal truncated or “ragged” variants of Aβ has been known for some time (see, e.g., Masters et al., 1985; Roher et al., 1993). The seminal paper by Takaomi Saido and coworkers (Saido et al., 1995) showed for the first time that Aβ3(pE) represents a dominant fraction of Aβ peptides in AD brain. N-terminal deletions in general enhance aggregation of β-amyloid peptides in vitro (Pike et al., 1995). Aβ3(pE) has a higher aggregation propensity (He and Barrow, 1999; Schilling et al., 2006), and stability (Kuo et al., 1998), and shows an increased toxicity compared to full-length Aβ (Russo et al., 2002). Schilling et al. have demonstrated that pyroglutamate-modified peptides display an up to 250-fold acceleration rate in the initial formation of Aβ aggregates (Schilling et al., 2006). Inhibition of enzymatic QC activity leads to significantly reduced Aβ3(pE) formation in vitro (Cynis et al., 2006).

    Maeda et al. have demonstrated that APP transgenic mouse models exhibit Aβ3(pE) levels to a different degree. Previously it has been shown that APP/PS1KI mice generate very high levels of Aβ3(pE) studied by 2D-gel electrophoresis (Casas et al., 2004) and immunostaining (Wirths et al., 2007a). Interestingly, this model elicits 50 percent CA1 neuron loss, axonopathy in brain and spinal cord (Wirths et al., 2007a), correlating well with the observed robust working memory and motor impairment (Wirths et al., 2007b).

    Overall, Maeda et al. have provided striking evidence that PiB retention is a consequence of Aβ3(pE) plaque deposition. This supports the notion that Aβ3(pE) is a major player in amyloid-driven pathology, which in my view deserves more attention in the future.

    References:

    . Massive CA1/2 neuronal loss with intraneuronal and N-terminal truncated Abeta42 accumulation in a novel Alzheimer transgenic model. Am J Pathol. 2004 Oct;165(4):1289-300. PubMed.

    . Inhibition of glutaminyl cyclase alters pyroglutamate formation in mammalian cells. Biochim Biophys Acta. 2006 Oct;1764(10):1618-25. PubMed.

    . The A beta 3-pyroglutamyl and 11-pyroglutamyl peptides found in senile plaque have greater beta-sheet forming and aggregation propensities in vitro than full-length A beta. Biochemistry. 1999 Aug 17;38(33):10871-7. PubMed.

    . Irreversible dimerization/tetramerization and post-translational modifications inhibit proteolytic degradation of A beta peptides of Alzheimer's disease. Biochim Biophys Acta. 1998 Apr 28;1406(3):291-8. PubMed.

    . Longitudinal, quantitative assessment of amyloid, neuroinflammation, and anti-amyloid treatment in a living mouse model of Alzheimer's disease enabled by positron emission tomography. J Neurosci. 2007 Oct 10;27(41):10957-68. PubMed.

    . Amyloid plaque core protein in Alzheimer disease and Down syndrome. Proc Natl Acad Sci U S A. 1985 Jun;82(12):4245-9. PubMed.

    . Amino-terminal deletions enhance aggregation of beta-amyloid peptides in vitro. J Biol Chem. 1995 Oct 13;270(41):23895-8. PubMed.

    . Structural alterations in the peptide backbone of beta-amyloid core protein may account for its deposition and stability in Alzheimer's disease. J Biol Chem. 1993 Feb 15;268(5):3072-83. PubMed.

    . Pyroglutamate-modified amyloid beta-peptides--AbetaN3(pE)--strongly affect cultured neuron and astrocyte survival. J Neurochem. 2002 Sep;82(6):1480-9. PubMed.

    . Dominant and differential deposition of distinct beta-amyloid peptide species, A beta N3(pE), in senile plaques. Neuron. 1995 Feb;14(2):457-66. PubMed.

    . On the seeding and oligomerization of pGlu-amyloid peptides (in vitro). Biochemistry. 2006 Oct 17;45(41):12393-9. PubMed.

    . Age-dependent axonal degeneration in an Alzheimer mouse model. Neurobiol Aging. 2007 Nov;28(11):1689-99. PubMed.

    . Deficits in working memory and motor performance in the APP/PS1ki mouse model for Alzheimer's disease. Neurobiol Aging. 2008 Jun;29(6):891-901. PubMed.

  4. The elegant report by Maeda and colleagues [1] shows that in vivo amyloid imaging with 11C-PIB in transgenic (Tg) mice is possible. After the initial in vivo studies with multiphoton microscopy showing binding of PIB to plaques in Tg mice [2], it was reported that 11C-PIB did not significantly bind to aggregated Aβ in Tg mice [3].

    The key to this groundbreaking report is the ability to inject mice with very high specific activity (SA) 11C-PIB. As Bill Klunk points out, what seems critical is not the amount of Aβ or the number of plaques but rather the amount of available binding sites, and their relative affinity, reflected in image contrast and the amount of non-specific binding. The 11C-PIB SA reported by Maeda were in excess of 7.9 Ci/μmol (or 5.4 Ci/μmol at the time of injection), much higher than the ones reported by Toyama (1.1-3.2 Ci/μmol) [3], or Klunk (>1 Ci/μmol) [4]. Not too many PET centers can achieve such high SA. To our knowledge, the only other group that has been able to show quantifiable images of 11C-PIB in Tg mice is the group led by Alexander Drzezga in Munich [5].

    On the other hand, a quick review of the PIB literature reveals that SA does not seem to be that crucial in 11C-PIB PET studies in Alzheimer patients or age-matched controls, with reported SA ranging from 0.68 Ci/μmol [6] to 4.3-4.5 Ci/μmol [7,8]. Nevertheless, a more detailed evaluation of the effects of high and low SA on 11C-PIB PET studies, as Maeda and colleagues suggest, may be warranted. The use of high SA 11C-PIB might also help better characterize Aβ deposition in asymptomatic control subjects [7,9].

    This study also sheds light into the kind of Aβ species bound by 11C-PIB in senile plaques, showing that it preferably binds to the N-terminal truncated Aβ species, more specifically the one truncated at position 3 (Aβ3[pE]). This is relevant because the accelerated formation of plaques seems to be associated with this Aβ3(pE)1-42(43) species. [10,11] The present study shows that 11C-PIB binding colocalized with Aβ3(pE)1-42(43), displaying a fivefold higher affinity than for Aβ1-42(43).

    Maeda and colleagues also evaluated the Aβ deposition in the mouse brain over time but, more importantly, the effect of intrahippocampal administration of passive anti-Aβ immunization, assessed by both 11C-PIB and 18F-fluoroethyl-DAA1106, a microglial activation radioligand, demonstrating the usefulness of this approach to evaluate and screen the effects of anti-Aβ treatment over time.

    This report highlights the need of using high SA 11C-PIB for microPET mice studies. On the other hand, these requirements might not be achievable in every PET center, precluding the application of this important approach to investigate the spatial and temporal pattern of Aβ deposition and monitor the effectiveness of novel anti-Aβ therapy.

    References:

    . Longitudinal, quantitative assessment of amyloid, neuroinflammation, and anti-amyloid treatment in a living mouse model of Alzheimer's disease enabled by positron emission tomography. J Neurosci. 2007 Oct 10;27(41):10957-68. PubMed.

    . Four-dimensional multiphoton imaging of brain entry, amyloid binding, and clearance of an amyloid-beta ligand in transgenic mice. Proc Natl Acad Sci U S A. 2003 Oct 14;100(21):12462-7. PubMed.

    . PET imaging of brain with the beta-amyloid probe, [11C]6-OH-BTA-1, in a transgenic mouse model of Alzheimer's disease. Eur J Nucl Med Mol Imaging. 2005 May;32(5):593-600. PubMed.

    . Binding of the positron emission tomography tracer Pittsburgh compound-B reflects the amount of amyloid-beta in Alzheimer's disease brain but not in transgenic mouse brain. J Neurosci. 2005 Nov 16;25(46):10598-606. PubMed.

    . Feasibility of in vivo amyloid plaque imaging in a transgenic mouse model of Alzheimer’s disease. J Nucl Med, 2007. 48(Suppl 2): p. 116P.

    . Imaging brain amyloid in Alzheimer's disease with Pittsburgh Compound-B. Ann Neurol. 2004 Mar;55(3):306-19. PubMed.

    . [11C]PIB in a nondemented population: potential antecedent marker of Alzheimer disease. Neurology. 2006 Aug 8;67(3):446-52. PubMed.

    . Kinetic modeling of amyloid binding in humans using PET imaging and Pittsburgh Compound-B. J Cereb Blood Flow Metab. 2005 Nov;25(11):1528-47. PubMed.

    . Imaging beta-amyloid burden in aging and dementia. Neurology. 2007 May 15;68(20):1718-25. PubMed.

    . Amyloid beta protein starting pyroglutamate at position 3 is a major component of the amyloid deposits in the Alzheimer's disease brain. Biochem Biophys Res Commun. 2000 Sep 24;276(2):422-7. PubMed.

    . On the seeding and oligomerization of pGlu-amyloid peptides (in vitro). Biochemistry. 2006 Oct 17;45(41):12393-9. PubMed.

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