This paper illustrates nicely how complex and dynamic the interactions between microglia and β amyloid are, and how relevant these interactions are to the pathogenesis of Alzheimer disease, at least in mouse models. The paper supports a role for microglia in clearing β amyloid and suggests that acute as well as chronic/long-term interactions of these cells with plaques are important and need to be investigated. I believe two important questions raised again by the paper (as well as by other papers) need to be addressed in future studies:

1. How do microglia accumulate in and around plaques? There is compelling data in the literature that support a migratory event involving recruitment to plaques, but perhaps also there is a small element of proliferation, though the question of proliferation needs to be further explored to be proven.

2. What are the molecular mechanisms by which microglia promote clearance of β amyloid and why do we still get plaques in spite of this microglial role?

View all comments by Joseph El Khoury

The paper by Bolmont and colleagues (2008) represents an elegant set of experiments designed to track microglia in a doubly-transgenic mouse model of AD. The authors crossed an Iba1-GFP transgenic (thereby labeling microglia green) with a co-injected APP/PS1 AD mouse and imaged cerebral vessels, microglia, and amyloid plaques using multi-photon microscopy, by way of a cranial window. They imaged these animals at short-time intervals (within minutes), and over longer time periods (from days to one month). In my view, there are a number of important take-home messages, and also a whole host of interesting questions raised by this work.

Importantly, the authors found in their “longer time period” imaging experiments that amyloid plaques are remarkably stable, as noted also by Joanna Jankowsky and David Borchelt using a tet-inducible AD mouse model (Jankowsky et al., 2005). This is an interesting result in and of itself, because an earlier view was that cerebral amyloid deposits were dynamic, coming and going based on changes in microenvironment. The authors have gone further by...  Read more

The paper by Bolmont and colleagues (2008) represents an elegant set of experiments designed to track microglia in a doubly-transgenic mouse model of AD. The authors crossed an Iba1-GFP transgenic (thereby labeling microglia green) with a co-injected APP/PS1 AD mouse and imaged cerebral vessels, microglia, and amyloid plaques using multi-photon microscopy, by way of a cranial window. They imaged these animals at short-time intervals (within minutes), and over longer time periods (from days to one month). In my view, there are a number of important take-home messages, and also a whole host of interesting questions raised by this work.

Importantly, the authors found in their “longer time period” imaging experiments that amyloid plaques are remarkably stable, as noted also by Joanna Jankowsky and David Borchelt using a tet-inducible AD mouse model (Jankowsky et al., 2005). This is an interesting result in and of itself, because an earlier view was that cerebral amyloid deposits were dynamic, coming and going based on changes in microenvironment. The authors have gone further by showing that microglia migrate toward amyloid deposits, and once they reach their destination, they remain there as permanent residents and enlarge their somas. While the authors do not report fixed tissue-immunostaining of this cohort of microglia with activation makers (e.g., CD11b/Mac-1, CD45, F4/80 Ag, etc.), by inference it is highly likely that these cells represent the “activated” microglia typically found in close vicinity to mature amyloid plaques, which are undergoing an anti-phagocytic, proinflammatory innate immune response (Town et al., 2005). This is further supported by the authors’ finding of reduced fine processes for microglia “on” plaques (probably representative of the activated, “amoeboid” morphology seen by others). The authors also found that microglia volume increased in proportion with amyloid plaque volume, providing strong evidence, in my view, that larger, “mature” amyloid plaques are more immunogenic than less mature plaques. This was most obvious when considering “medium-sized” versus “large” amyloid plaques, where the latter showed a 225 percent increase in microglia volume.

I find most intriguing the authors’ finding that microglia surrounding amyloid plaques displayed puncta of the dye used for Aβ imaging, which generally persisted throughout the duration of the experiments. Pioneering early reports from Henry Wisniewski and Jerzy Wegiel demonstrated at the ultrastructural level that microglia around plaques fail to internalize amyloid fibrils (Wisniewski et al., 1989; Wegiel and Wisniewski, 1990; Wisniewski and Wegiel, 1994). More recent reports (Stalder et al., 1999; 2001) have further highlighted that fibrillar amyloid is not present within microglia, seemingly at odds with the authors’ finding. However, as the authors rightly point out, differences in methodology (not the least of which is in-vivo imaging versus postmortem analysis of tissue sections) may account for this. Assuming this result is robust, it is interesting that these puncta generally remained visible throughout the course of the experiment, suggesting that even if microglia are capable of internalizing amyloid deposits in vivo, they are not efficient amyloid degraders. This begs the critically important question of how to promote efficient microglia-mediated clearance of amyloid plaques as a potential therapeutic modality, which is something that we are also intensely interested in (Town et al., 2005).

A number of interesting questions arise from this paper. Throughout the course of their long-term imaging experiments, did the authors detect new, rapidly forming plaques as reported recently by Brad Hyman’s group (Meyer-Luehmann et al., 2008)? If so, did microglia migrate to these plaques with differing kinetics from already-formed ones? Also, the authors mention the important point that they cannot discriminate between newly emigrating blood-derived monocytes/macrophages and resident microglial cells; however, they do report that, while small plaques increased in size by 84 percent, large plaques actually decreased in size by 12 percent. If not due to measurement error, one possibility is that the reduced size of large plaques could be due to infiltrating monocytes/macrophages, which may be more tuned to remove amyloid plaques.

References:
Jankowsky JL, Slunt HH, Gonzales V, Savonenko AV, Wen JC, Jenkins NA, Copeland NG, Younkin LH, Lester HA, Younkin SG, Borchelt DR. Persistent amyloidosis following suppression of Abeta production in a transgenic model of Alzheimer disease. PLoS Med. 2005 Dec;2(12):e355. Epub 2005 Nov 15. Abstract

Meyer-Luehmann M, Spires-Jones TL, Prada C, Garcia-Alloza M, de Calignon A, Rozkalne A, Koenigsknecht-Talboo J, Holtzman DM, Bacskai BJ, Hyman BT. 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. Abstract

Stalder M, Phinney A, Probst A, Sommer B, Staufenbiel M, Jucker M. Association of microglia with amyloid plaques in brains of APP23 transgenic mice. Am J Pathol. 1999 Jun;154(6):1673-84. Abstract

Stalder M, Deller T, Staufenbiel M, Jucker M. 3D-Reconstruction of microglia and amyloid in APP23 transgenic mice: no evidence of intracellular amyloid. Neurobiol Aging. 2001 May-Jun;22(3):427-34. Abstract

Town T, Nikolic V, Tan J. The microglial "activation" continuum: from innate to adaptive responses. J Neuroinflammation. 2005 Oct 31;2:24. Abstract

Wisniewski HM, Wegiel J, Wang KC, Kujawa M, Lach B. Ultrastructural studies of the cells forming amyloid fibers in classical plaques. Can J Neurol Sci. 1989 Nov;16(4 Suppl):535-42. Abstract

Wegiel J, Wisniewski HM. The complex of microglial cells and amyloid star in three-dimensional reconstruction. Acta Neuropathol. 1990;81(2):116-24. Abstract

Wisniewski HM, Wegiel J. The role of microglia in amyloid fibril formation. Neuropathol Appl Neurobiol. 1994 Apr;20(2):192-4. Abstract

View all comments by Terrence Town

This is a fascinating paper from the group of Mathias Jucker and Michael Calhoun studying in vivo interaction of amyloid plaques and their most intriguing and prominent cellular component—the microglia. Employing two-photon imaging of GFP-labeled microglia, methoxy-X04-labeled plaques, and triangulating on the study areas for repeated measures with the help of dextran conjugate-labeled blood vessels, the authors support and extend previous observations (Davalos et al., 2005; Nimmerjahn et al., 2005; Meyer-Luehmann et al., 2008) of a highly dynamic role of microglia in conducting brain surveillance. Like a true vigilante, supposedly “resting” microglia continually patrol their microenvironment with extremely motile ramified cellular processes. At the first contact of amyloid plaques, they jump on-scene and try to clear the plaques and, expectedly, do some collateral damage to the surrounding environment.

One of the many reasons I find this study fascinating is that it tries to “assess” not only how many microglia arrive at the sites of plaques, but also how fast they do so, and...  Read more

This is a fascinating paper from the group of Mathias Jucker and Michael Calhoun studying in vivo interaction of amyloid plaques and their most intriguing and prominent cellular component—the microglia. Employing two-photon imaging of GFP-labeled microglia, methoxy-X04-labeled plaques, and triangulating on the study areas for repeated measures with the help of dextran conjugate-labeled blood vessels, the authors support and extend previous observations (Davalos et al., 2005; Nimmerjahn et al., 2005; Meyer-Luehmann et al., 2008) of a highly dynamic role of microglia in conducting brain surveillance. Like a true vigilante, supposedly “resting” microglia continually patrol their microenvironment with extremely motile ramified cellular processes. At the first contact of amyloid plaques, they jump on-scene and try to clear the plaques and, expectedly, do some collateral damage to the surrounding environment.

One of the many reasons I find this study fascinating is that it tries to “assess” not only how many microglia arrive at the sites of plaques, but also how fast they do so, and how successfully they limit or resolve these plaques. Though not quantitatively, the study also tries to find in vivo evidence of Aβ phagocytosis by microglia.

For instance, on the high-resolution time-lapse images acquired over minutes to hours or days, about half of the studied microglia were shown to migrate to the proximity of the amyloid plaques within 24-48 hours. These data seem to differ from some other studies where only microglial processes were observed to be dynamic but not their somas (Davalos et al., 2005; Nimmerjahn et al., 2005). However, these prior studies are based on brain injury models, and the impact of such injuries might be different from less acute but difficult-to-heal Aβ plaques.

Movement of microglial cell bodies to the site of amyloid plaques has also been observed to occur within 24 hours in a recently published study (Meyer-Luehmann et al., 2008). It is possible that a tighter and more “stable” cell soma-Aβ contact, as shown in the current paper, is necessary for attempted walling off and/or phagocytosis by microglia. In contrast to Meyer-Luehmann et al. (2008), but similar to several previous histology-based studies, the authors also showed globular intracellular methoxy-X04 labeling within microglia, which on parallel histology studies colocalized with the lysosomal marker lamp-1. However, whether these mononuclear phagocytes are bone marrow (BM)-derived or resident microglia remains an open question. BM-derived macrophages have been recently shown to infiltrate rodent brain in response to injected Aβ40 and Aβ42, and they were further able to phagocytose amyloid, while resident microglia did not appear to do (Simard et al., 2006).

How about the age-old question, Is microglia-Aβ interaction harmful or beneficial? The current study and similar studies showing a role of microglia in walling off the injured areas and in limiting plaque growth clearly point towards a beneficial effect (Simard et al., 2006; Bolmont et al., 2008). Deficiency of Ccr2, a chemokine receptor expressed on microglia, accelerating early disease progression in rodents, also supports these data (El Khoury et al., 2007). But we have to keep in mind that the rodent data have still to be correctly translated to humans. For instance, controlled human clinical trials have repeatedly shown less robust effects of anti-inflammatory drugs in contrast to their fantastic beneficial effects on mouse models. Some of the issues raised in the current paper, such as dynamics of plaque growth due to soluble Aβ, is already one factor that would be different in humans. Thus, while not all is answered, the in vivo approach employed in some of these elegant papers is definitely progress in the right direction.

References:
Davalos D, Grutzendler J, Yang G, Kim JV, Zuo Y, Jung S, Littman DR, Dustin ML, Gan WB. ATP mediates rapid microglial response to local brain injury in vivo. Nat Neurosci. 2005 Jun;8(6):752-8. Abstract

El Khoury J, Toft M, Hickman SE, Means TK, Terada K, Geula C, Luster AD. Ccr2 deficiency impairs microglial accumulation and accelerates progression of Alzheimer-like disease. Nat Med. 2007 Apr;13(4):432-8. Abstract

Meyer-Luehmann M, Spires-Jones TL, Prada C, Garcia-Alloza M, de Calignon A, Rozkalne A, Koenigsknecht-Talboo J, Holtzman DM, Bacskai BJ, Hyman BT. 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. Abstract

Nimmerjahn A, Kirchhoff F, Helmchen F. Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo. Science. 2005 May 27;308(5726):1314-8. Abstract

Simard AR, Soulet D, Gowing G, Julien JP, Rivest S. Bone marrow-derived microglia play a critical role in restricting senile plaque formation in Alzheimer's disease. Neuron. 2006 Feb 16;49(4):489-502. Abstract

View all comments by Samir Kumar-Singh

This paper by the group of Michael Calhoun confirms, in vivo, a role for microglia in amyloid plaque maintenance. The authors succeeded in monitoring live changes in plaque size, and correlate them to changes in number and appearance of “on site” microglial cells. Interestingly, they describe microglial uptake of the amyloid-β fibrils bound to the methoxy-X04 dye. This is an unexpected result that contradicts previous reports (Stalder et al., 2001). However, if indeed microglial cells can phagocytose Aβ fibrils, why are these cells not able to completely disassemble or clear Aβ plaques? Instead, microglial cells seem to just guarantee a stable plaque size after their initial acute growth, as previously suggested by Meyer-Luehmann et al. (2008). Animal species differences should be also taken into account, since findings for human microglia cells can contrast with the one for murine microglial cells (Blasi et al., 1995).

Overall, this paper opens the door to understanding the mechanism with which microglia would control the size of amyloid-β plaques and therefore to new...  Read more

This paper by the group of Michael Calhoun confirms, in vivo, a role for microglia in amyloid plaque maintenance. The authors succeeded in monitoring live changes in plaque size, and correlate them to changes in number and appearance of “on site” microglial cells. Interestingly, they describe microglial uptake of the amyloid-β fibrils bound to the methoxy-X04 dye. This is an unexpected result that contradicts previous reports (Stalder et al., 2001). However, if indeed microglial cells can phagocytose Aβ fibrils, why are these cells not able to completely disassemble or clear Aβ plaques? Instead, microglial cells seem to just guarantee a stable plaque size after their initial acute growth, as previously suggested by Meyer-Luehmann et al. (2008). Animal species differences should be also taken into account, since findings for human microglia cells can contrast with the one for murine microglial cells (Blasi et al., 1995).

Overall, this paper opens the door to understanding the mechanism with which microglia would control the size of amyloid-β plaques and therefore to new possible therapeutic approaches.

References:
Blasi E, Barluzzi R, Mazzolla R, Tancini B, Saleppico S, Puliti M, Pitzurra L, Bistoni F. Role of nitric oxide and melanogenesis in the accomplishment of anticryptococcal activity by the BV-2 microglial cell line. J Neuroimmunol. 1995 Apr;58(1):111-6. Abstract

Meyer-Luehmann M, Spires-Jones TL, Prada C, Garcia-Alloza M, de Calignon A, Rozkalne A, Koenigsknecht-Talboo J, Holtzman DM, Bacskai BJ, Hyman BT. 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. Abstract

Stalder M, Deller T, Staufenbiel M, Jucker M. 3D-Reconstruction of microglia and amyloid in APP23 transgenic mice: no evidence of intracellular amyloid. Neurobiol Aging. 2001 May-Jun;22(3):427-34. Abstract

View all comments by Barbara Calabrese

This research design could provide a means of exploring curcumin's potential role as a facilitator of microglial phagocytosis and degradation of amyloid plaque.

View all comments by Stephen Staten

As a graduate student who reviewed this subject in great detail for a journal club (see Meyer-Luehmann et al., 2008 and Yan et al., 2009), I am surprised at some of the opinions presented here after these most recent papers on plaque dynamics (Hefendehl et al., 2011; Burgold et al., 2010), which I think are interesting and thorough examinations of plaque growth in vivo. In contrast, when reviewing the initial paper on this topic from the Hyman Lab (Meyer-Luehmann et al., 2008), it became apparent to me and the people with whom I discussed it that the reason why they saw very rapid plaque appearance and no further plaque growth within 14 days was because of an artifact of incomplete dye labeling. If one inspects in detail Figure 1 in their paper, one can see that the plaque that “appeared” after 24 hours of dye injection was really present even before...  Read more

As a graduate student who reviewed this subject in great detail for a journal club (see Meyer-Luehmann et al., 2008 and Yan et al., 2009), I am surprised at some of the opinions presented here after these most recent papers on plaque dynamics (Hefendehl et al., 2011; Burgold et al., 2010), which I think are interesting and thorough examinations of plaque growth in vivo. In contrast, when reviewing the initial paper on this topic from the Hyman Lab (Meyer-Luehmann et al., 2008), it became apparent to me and the people with whom I discussed it that the reason why they saw very rapid plaque appearance and no further plaque growth within 14 days was because of an artifact of incomplete dye labeling. If one inspects in detail Figure 1 in their paper, one can see that the plaque that “appeared” after 24 hours of dye injection was really present even before (just poorly labeled). Consistent with this, the adjacent large plaque seen in the same image underwent a very marked increase in dye labeling within this same interval. This is almost certain to be explained by ongoing dye labeling.

Interestingly, in this same figure, they present their data of all new plaques observed, and coincidentally they all appeared within one day of the first dye injection. This again is consistent with an artifact in which dye labeling is incomplete after 24 hours of initial dye injection. The appearance of a plaque at around 24 hours just reflects the ongoing dye labeling. Incomplete labeling also explains why they did not see any new plaques appearing at any time other than after the first day of dye injection. In my opinion, their paper remains at odds with these more recent papers in the Journal of Neuroscience and Acta Neuropathologica, which show no rapid plaque appearance and report continuous plaque growth over much longer intervals. The lack of growth seen in the Hyman paper is likely to be related to neuroinflammation induced by their imaging procedure as previously demonstrated (Yan et al., 2009).

References:
Meyer-Luehmann M, Spires-Jones TL, Prada C, Garcia-Alloza M, de Calignon A, Rozkalne A, Koenigsknecht-Talboo J, Holtzman DM, Bacskai BJ, Hyman BT. 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. Abstract

Hefendehl JK, Wegenast-Braun BM, Liebig C, Eicke D, Milford D, Calhoun ME, Kohsaka S, Eichner M, Jucker M. Long-term in vivo imaging of beta-amyloid plaque appearance and growth in a mouse model of cerebral beta-amyloidosis. J. Neurosci. 2011;31(2):624-629. Abstract

Burgold S, Bittner T, Dorostkar MM, Kieser D, Fuhrmann M, Mitteregger G, Kretzschmar H, Schmidt B, Herms J. In vivo multiphoton imaging reveals gradual growth of newborn amyloid plaques over weeks. Acta Neuropathol. 2010 Dec 6. Abstract

Christie RH, Bacskai BJ, Zipfel WR, Williams RM, Kajdasz ST, Webb WW, Hyman BT. Growth arrest of individual senile plaques in a model of Alzheimer's disease observed by in vivo multiphoton microscopy. J Neurosci. 2001 Feb 1;21(3):858-64. Abstract

View all comments by Jason Frommer

Several papers now have used multiphoton imaging to monitor plaques over time in AD transgenic models (Hefendehl et al., 2011; Burgold et al., 2010; Yan et al., 2009), following on the initial work we published in 2001 (Christie et al., 2001). Over the years we have imaged thousands of plaques using either “thin skull” or “coverslip” approaches in three different APP or APP/PS1 overexpressing models. The new papers, emerging from analogous work at Washington University and in Germany, show similar approaches to dissect the natural history of plaques in living animals.

Overall, there is general concurrence in our observations. It is obvious that animals initially have no plaques, then many months later have many plaques. What happens in between? We found that plaques form surprisingly quickly, then reach a near maximal size within days. The other groups, using slightly different models and...  Read more

Several papers now have used multiphoton imaging to monitor plaques over time in AD transgenic models (Hefendehl et al., 2011; Burgold et al., 2010; Yan et al., 2009), following on the initial work we published in 2001 (Christie et al., 2001). Over the years we have imaged thousands of plaques using either “thin skull” or “coverslip” approaches in three different APP or APP/PS1 overexpressing models. The new papers, emerging from analogous work at Washington University and in Germany, show similar approaches to dissect the natural history of plaques in living animals.

Overall, there is general concurrence in our observations. It is obvious that animals initially have no plaques, then many months later have many plaques. What happens in between? We found that plaques form surprisingly quickly, then reach a near maximal size within days. The other groups, using slightly different models and methods, found that plaques form and then may well continue to grow initially for some time, then reach a plateau where growth ceases. That growth ultimately ceases is obvious—otherwise there would be one large plaque in the brains of elderly mice, and, of course, that is not the case. In fact, postmortem analysis of plaque size distribution reveals no change in the average size of plaques or in the distribution of sizes regardless of age.

Why are there any differences in the observations regarding the slope of the growth of plaques in animal models? Any number of technical issues—ranging from mouse variability to differences in imaging techniques—might help explain the discrepancies. We have measured cross-sectional areas because of the increased resolution of images in the X-Y plane, while other groups use a full Z stack and estimate volume, essentially trading the increased information in the Z stack for the increased uncertainties of the measurements at the top and bottom (given relatively poor Z resolution compared to X-Y resolution in multiphoton optics). Different surgical procedures, different ways of administering dyes, different software packages, or even different optics might impact the subtle analysis of these high-resolution images.

However, the important point is whether any of these observations accurately model what happens in Alzheimer’s disease itself. From this point of view, we have recently completed an analysis of the temporal neocortex of 92 individuals with Alzheimer's disease, and 16 controls, ranging in duration of dementia from six months to almost 20 years. Of course, this is a postmortem histological analysis, so that longitudinal imaging of individual plaques is not possible. Nonetheless, if plaques dramatically grew with increasing duration of illness, we would expect to see evidence of that in the size distribution of either the thioflavin S core or the anti-Aβ immunostained deposits. We found only the most subtle changes over time, with an increase in plaque size over 20 years of ~2 percent per year. We conclude that dramatic continued plaque growth is unlikely to be a central feature of Alzheimer's disease progression, although the conundrum still remains as to why plaques form in the first place, grow to their rather large size, and then presumably ultimately reach a plateau where further growth is inhibited. It may be that careful analysis of what impacts the rate of growth, or of the phenomena that occur after plaques stabilize, will help provide insight. We hope that in vivo multiphoton longitudinal imaging of animal models will continue to help point towards answers to these sorts of questions.

View all comments by Brian Bacskai
View all comments by Bradley Hyman