. Rapid appearance and local toxicity of amyloid-beta plaques in a mouse model of Alzheimer's disease. Nature. 2008 Feb 7;451(7179):720-4. PubMed.

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  1. This is another state-of-the-art paper by the group active on in vivo two-photon imaging on mouse models of amyloidogenesis, and it gives important clues for Alzheimer disease pathogenesis. The paper shows, for the first time, that dense plaques in mouse models reach their maximum size in about a day and thereafter maintain a status quo. This does not follow the simple, size-dependent law of mass action, as even small plaques do not grow any further. This is a most amazing finding and abrogates all prior preconceived notions that plaques grow slowly over life and that given time, all plaques would reach a maximum size. Importantly, the quick growth of dense plaques suggests that dense plaques grow not only with Aβ monomer addition, but perhaps also by capturing oligomeric intermediates at the fiber ends, as shown earlier for prion proteins (Serio et al., 2000; Collins et al., 2004).

    Why dense plaques stop growing suddenly is just as intriguing. Quick recruitment of macrophages at sites of dense plaque formation, as shown here, could be one mechanism, but the provided images do not show a complete walling off. This suggests that other, less simple mechanisms are at play, including local Aβ production and trafficking.

    A recent, interesting study in a mouse model of amyloidogenesis showed that FAD APP mutations cause axonal trafficking defects and that that, in turn, stimulates the proteolytic processing of APP and generation of Aβ (Stokin et al., 2005). This fit well with development of dystrophic neurites (DNs) in these mice, preceding plaques by more than a year (Stokin et al., 2005). Meyer-Luehmann and colleagues, studying the temporal relation between rapidly growing plaques and DNs, showed that although DNs were observed in plaque-free areas, DNs were more pronounced near plaques. Conversely, DNs in the plaque-free areas did not seem to cause dense plaques, and mice did not deposit diffuse plaques at this age. It would be worth studying this in more detail over a shorter time window, as DNs were also observed to change morphologies and even resolve, leaving open the possibility that diffuse or pre-diffuse plaques are formed but are quickly turned over. Moreover, the current resolution of this technique does not permit visualization of small, dense plaques, and at times it leaves the reader guessing whether some of the punctate blue staining might potentially be “sub-microscopic” dense plaques (for instance, boxed areas in Figure 1). Also, sometimes the capillary network is strangely absent, for instance, some panels of Figure 1 representing APPswe/PS1d9xYFP mice, where the plaque-vessel relationship has been alluded to on a small sample size.

    Clearly, more work is needed before we can have all the answers. Meanwhile, despite some of its limitations, in vivo multiphoton imaging remains a valuable technique. We hope to see more results coming out from this technology, especially with higher objectives, use of confocal settings, and perhaps a shorter time lapse, even though that would make it even more labor-intensive. Parallel detailed histological analysis including ultrastructural microscopy should make it even more interesting.

    References:

    . Mechanism of prion propagation: amyloid growth occurs by monomer addition. PLoS Biol. 2004 Oct;2(10):e321. PubMed.

    . Rapid appearance and local toxicity of amyloid-beta plaques in a mouse model of Alzheimer's disease. Nature. 2008 Feb 7;451(7179):720-4. PubMed.

    . Nucleated conformational conversion and the replication of conformational information by a prion determinant. Science. 2000 Aug 25;289(5483):1317-21. PubMed.

    . Dendritic spine abnormalities in amyloid precursor protein transgenic mice demonstrated by gene transfer and intravital multiphoton microscopy. J Neurosci. 2005 Aug 3;25(31):7278-87. PubMed.

    . Axonopathy and transport deficits early in the pathogenesis of Alzheimer's disease. Science. 2005 Feb 25;307(5713):1282-8. PubMed.

  2. Meyer-Luehmann et al. provide a spectacular and informative high-tech view of the kinetics of amyloid formation and its potential consequences in a mouse model of Alzheimer disease. Although some may regard their findings as contradictory to the idea that early transport defects may play a role in neuronal dysfunction and in the enhancement of amyloid formation in Alzheimer disease, I do not see this study as being in conflict with those ideas. There are two major points:

    1. In addition to the experiments we reported (Stokin et al., 2005), there are a number of previous studies (cited in Stokin et al., 2005) that find significant axonal dystrophies, which may be indicative of defects in axonal transport, prior to amyloid deposition as well as in regions of the brain that lack amyloid deposition. A related issue is that the experiments of Meyer-Luehmann et al. primarily focus on cortical regions, while many of the experiments in Stokin et al. examined basal forebrain cholinergic axons, which are long projection axons in regions distinct from the cortical regions imaged in the present paper.

    2. A number of papers (Torroja et al., 1999; Gunawardena et al., 2001; Salehi et al., 2006; Pigino et al., 2003) report that APP overexpression and presenilin mutations, both of which can cause Alzheimer disease, can induce serious abnormalities in axonal transport in the absence of plaque formation or human Aβ. Again, these findings and those of Meyer-Luehmann et al. are not in conflict and may in some sense be complementary.

    A possibly useful way to think about this collection of observations is by asking two questions: 1) Are amyloid plaques completely benign? 2) Are amyloid plaques or their constituents sufficient to induce all of the biochemical and cellular malfunctions associated with Alzheimer disease? The answer to the first question is almost surely no—it is hard to imagine that massive numbers of amyloid plaques in the brain would not interfere with neuronal function. Indeed, there is ample documentary evidence that amyloid constituents can be neurotoxic to varying degrees. The answer to the second question is far from clear. APP processing clearly generates fragments other than Aβ, and may influence other biological activities of APP and neurons. It may be that a productive additional approach to this question is by trying to understand the relative toxicity of Aβ to neurons compared to toxicity caused by other environmental influences, mutations, pathways, or polymorphisms that all may reduce or impair the supply of critical materials to synapses by the axonal transport machinery.

    References:

    . Axonopathy and transport deficits early in the pathogenesis of Alzheimer's disease. Science. 2005 Feb 25;307(5713):1282-8. PubMed.

    . Neuronal overexpression of APPL, the Drosophila homologue of the amyloid precursor protein (APP), disrupts axonal transport. Curr Biol. 1999 May 6;9(9):489-92. PubMed.

    . Disruption of axonal transport and neuronal viability by amyloid precursor protein mutations in Drosophila. Neuron. 2001 Nov 8;32(3):389-401. PubMed.

    . Increased App expression in a mouse model of Down's syndrome disrupts NGF transport and causes cholinergic neuron degeneration. Neuron. 2006 Jul 6;51(1):29-42. PubMed.

    . Alzheimer's presenilin 1 mutations impair kinesin-based axonal transport. J Neurosci. 2003 Jun 1;23(11):4499-508. PubMed.

  3. This is a beautiful paper showing plaque growth in vivo. I cordially disagree on one point: the authors state that the speed of an individual plaque's growth is surprising because of prior in vitro studies of protein aggregation showing a slow, time-dependent course. The discussion appears to suggest that the appearance of a plaque within a day or two does not fit in with data on nucleation-dependent polymerization (Jarrett and Lansbury, 1993). In fact, the observations in this paper are reminiscent of seeded crystal growth. Live multiphoton imaging cannot yet visualize the prior accumulation of Aβ or the nucleation event, but once nucleation happens, growth should be very fast. The rate of growth measured in this study is exactly what the nucleation model would predict. It is gratifying to see in vivo.

    References:

    . Seeding "one-dimensional crystallization" of amyloid: a pathogenic mechanism in Alzheimer's disease and scrapie?. Cell. 1993 Jun 18;73(6):1055-8. PubMed.

  4. This paper is intriguing, to say the least. The authors succeeded in monitoring in vivo the formation of dense-core plaques. Surprisingly, they observed that across different mouse models of Alzheimer disease, plaques formed quite rapidly (24 hours) but rarely. One of the most interesting observations of the paper is the temporal relation between rapid dense-core plaque appearance, microglial recruitment, and neuritic changes. Morphological changes of neurites never preceded plaque appearance and/or microglia migration towards the site of the newly formed plaque. Microglia did not seem to either facilitate or clear plaques, suggesting that they may participate in stabilizing plaque size after their initial acute growth.

    If we consider that, in recent years, soluble Aβ oligomers rather than Aβ fibrils in plaques have come to be seen as the “real bad guys” (Walsh and Selkoe, 2007), these new findings raise nearly as many questions as they answer. For example, are the neuritic alterations described in this paper induced by soluble forms of amyloid-β or by plaques themselves? If the former, why would soluble Aβ oligomers be responsible for dysmorphic neurites only when released by the plaques, but not as precursors of the same plaques? And even if we assume that these newly formed plaques function just as a local source of highly concentrated soluble Aβ, then why would dystrophic neurites take several days to appear? Faster morphological, functional, and behavioral alterations induced by soluble Aβ oligomers have been described both in cultured neurons (Calabrese et al., 2007), slices (Klyubin et al., 2005; Shankar et al., 2007), and in living rats (Cleary et al., 2005).

    To prove a direct role for plaques in inducing morphological changes, one would have to show that a selectively induced disassembly of the dense-core plaques would result in microglia dispersion and restoration of normal neurites. Recently, Martins et al. (2008) have shown that biologically relevant lipids, including lipid extracts from brain, can revert inert Aβ amyloid fibrils into neurotoxic protofibrils. Microglia could use a similar mechanism to control the size of amyloid-β plaques.

    Finally, the biggest challenge will be to identify the predictors for such sudden and local appearance of amyloid-β plaques so that eventually their formation can be prevented.

    References:

    . Rapid, concurrent alterations in pre- and postsynaptic structure induced by naturally-secreted amyloid-beta protein. Mol Cell Neurosci. 2007 Jun;35(2):183-93. PubMed.

    . Natural oligomers of the amyloid-beta protein specifically disrupt cognitive function. Nat Neurosci. 2005 Jan;8(1):79-84. PubMed.

    . Amyloid beta protein immunotherapy neutralizes Abeta oligomers that disrupt synaptic plasticity in vivo. Nat Med. 2005 May;11(5):556-61. PubMed.

    . Lipids revert inert Abeta amyloid fibrils to neurotoxic protofibrils that affect learning in mice. EMBO J. 2008 Jan 9;27(1):224-33. PubMed.

    . Natural oligomers of the Alzheimer amyloid-beta protein induce reversible synapse loss by modulating an NMDA-type glutamate receptor-dependent signaling pathway. J Neurosci. 2007 Mar 14;27(11):2866-75. PubMed.

    . A beta oligomers - a decade of discovery. J Neurochem. 2007 Jun;101(5):1172-84. PubMed.

  5. I agree with Peter Lansbury. This is a beautiful piece of research, though the rapidity with which distinct plaques could be visualized is not surprising to those of us who have studied the deposition of beta-amyloid in near-physiological milieu in vitro. Neither should we be surprised that plaque formation was coincident with both an immune/inflammatory response and damage to the environment adjacent to the plaque.

    I picked up my electronic copy of Nature early on the 7th and was able to present these results to medical students at 10.00 a.m. that morning. Congratulations are due to the authors for making simple what must have been extremely difficult to achieve.

  6. This captures the extraordinarily rapid growth of Aβ plaques in real time using sophisticated longitudinal multiphoton microscopy. Microglial cells are “caught in the act” of activation and recruitment. The data make for compelling watching, almost like witnessing a crime.

    What does this data suggest in terms of AD patients being treated with drugs to lower Aβ and to inhibit plaque formation? Do these new findings suggest that if drug treatment is discontinued, plaque growth and neurite dystrophy would recommence within days?

    In addition, this study reminds me of a previous finding, incredible though it seemed at the time, that AD model mice demonstrated immediate cognitive improvement after passive anti-Aβ immunization (Dodart et al., 2002; Kotilinek et al., 2002). It would be informative if the in-vivo microscopy could be used after immunization to observe microglial activation and recruitment, since the technique has already been used to monitor neurite dystrophy following passive (or active) immunization (Brendza et al., 2005; Lombardo et al., 2003). The technique could be very useful to determine the relationship between microglial activation and neurite dystrophy in response to changing levels of plaque Aβ.

    References:

    . Immunization reverses memory deficits without reducing brain Abeta burden in Alzheimer's disease model. Nat Neurosci. 2002 May;5(5):452-7. PubMed.

    . Reversible memory loss in a mouse transgenic model of Alzheimer's disease. J Neurosci. 2002 Aug 1;22(15):6331-5. PubMed.

    . Anti-Abeta antibody treatment promotes the rapid recovery of amyloid-associated neuritic dystrophy in PDAPP transgenic mice. J Clin Invest. 2005 Feb;115(2):428-33. PubMed.

    . Amyloid-beta antibody treatment leads to rapid normalization of plaque-induced neuritic alterations. J Neurosci. 2003 Nov 26;23(34):10879-83. PubMed.

  7. Using sophisticated life imaging techniques, Meyer-Luehmann and colleagues looked at plaque formation in the mouse brain in real time. The paper shows exciting results demonstrating that plaque deposition is very fast and that many of the pathological changes associated with plaques do not precede, but follow deposition, suggesting a cause-consequence relationship. The overall picture emerging is that plaques rapidly crystallize out of solution. Obviously, as interesting as it is, this work does not address the question of the mechanism of toxicity, neither of what determines the dynamics and the rapid precipitation of plaques in the brain.

    In our hands amyloid fibrils, as they are supposed to be present in amyloid plaques, display very little toxicity as such. Only when these mature fibrils become resolubilized, for instance, by lipids, do we generate what we called backward oligomers, which exert severe toxicity in neuronal culture and in brain of living animals (Martins and Kuperstein et al., 2008). There is also a recent study by Lesne and colleagues (2008) in plaque-bearing aged mice showing that memory impairment depends on soluble oligomers and not on the plaques. Thus, we remain with the question of whether the precipitated plaques seen by Meyer-Luehmann and colleagues directly cause toxicity, or whether around these plaques soluble oligomers or protofibrils are generated in dynamic equilibrium with the suddenly appearing plaques. If such an equilibrium exists, one would, however, have anticipated that plaques would appear and disappear, while these authors find that they are rather stable once they are formed. On the other hand, this exchange between soluble species and plaques may involve a limited number of deposited fibrils in the plaques that possibly does not change the structure of the plaque core once deposited, but is sufficient to produce toxic backward oligomers. Therefore, follow-up study of the presence of soluble oligomers, and of the dynamics of their appearance around the plaque, would be very interesting.

    One of the main questions raised by several commentators is, If soluble oligomers are the major source of neurotoxicity, then why does neurodystrophy appear after the deposition of amyloid and not before? Conclusions cannot be made in this regard. There is the whole problem of kinetics and concentration-dependent factors that have to be taken into account. It could be that the threshold for toxicity is close to the threshold for amyloid precipitation, and that only close to the plaques sufficient oligomers are present to cause toxicity. The final neurodystrophy effect seen 5 days after the plaque “birthday” could also be simply the result of a relatively slow cumulative effect of protofibrils/oligomers present before deposition and maintained afterwards by “backward” toxic species released from this newborn plaque.

    The question about stability of plaques as a function of time is also very intriguing. It would be informative to compare stability of fibrils in old and newborn plaques: the current study shows only the net result. In addition, it could indeed be that the microglia control the plaque size (Herber et al., 2007). In this regard, it becomes an interesting question to ask whether microglia are then protective, ensuring clearance of soluble toxic species, or whether they accelerate (backward) oligomer release.

    References:

    . Lipids revert inert Abeta amyloid fibrils to neurotoxic protofibrils that affect learning in mice. EMBO J. 2008 Jan 9;27(1):224-33. PubMed.

    . Plaque-bearing mice with reduced levels of oligomeric amyloid-beta assemblies have intact memory function. Neuroscience. 2008 Feb 6;151(3):745-9. PubMed.

    . Microglial activation is required for Abeta clearance after intracranial injection of lipopolysaccharide in APP transgenic mice. J Neuroimmune Pharmacol. 2007 Jun;2(2):222-31. PubMed.