Plaque Growth Gradual, Slowed by Gliosis and Aβ Removal

Imaging the brains of living mice through ultra-thin bone windows, scientists have demonstrated in vivo that amyloid plaques form and reach their full size over a period of weeks. This study, from Jin-Moo Lee and colleagues at Washington University School of Medicine, St. Louis, Missouri, adds a new, partly contradictory perspective to a recent high-profile paper on the dynamics of plaque formation. Lee’s team also provides evidence that plaque growth can be slowed by local gliosis and by small, timely reductions of soluble extracellular Aβ.

The question of how plaques form and grow is one that only a handful of labs are equipped to tackle head-on—that is, using in vivo multiphoton microscopy to watch the process unfold in real time in living animals. Brad Hyman, Brian Bacskai, and others at Massachusetts General Hospital, Charlestown, applied this technique in three mouse models of Alzheimer disease and observed something astonishing: by and large, the plaques they saw were cropping up overnight and hardly growing beyond that (Meyer-Luehmann et al., 2008 and ARF related news story). “I think it was assumed all along (that plaques grow), but when the Hyman paper came out, it was really questioned,” Lee said in an interview with ARF.

Lee and coworkers had been trying to address the same issue with a related approach that is less invasive but more technically demanding than the one used by the MGH group. Both labs used multiphoton microscopy; they differed in how they prepared the cranial windows for visualizing plaques. Hyman and colleagues imaged plaques through a glass slide affixed over a small hole in the mouse’s head. Lee’s team prepared the windows by painstakingly shaving away the skull “to the point where it’s so thin, it’s transparent,” he said. “You’re not opening up the skull, so you’re observing what is presumed to be intact brain without disrupting that integrity.”

To characterize plaque formation and growth, first author Ping Yan and colleagues imaged plaques across several time intervals in six- or 10-month-old APP/PS1 transgenic mice. Plaques in the older mice grew minimally, even over the longest period of observation, i.e., 90 days. However, one-fifth of the plaques in the six-month-old mice at least doubled in size within one week, and over a 28-day period, 62 percent had grown to that extent, some much more. “They saw plaques growing over periods of weeks, whereas we found that they appear within 24 hours,” said Bacskai, a coauthor on the earlier study using the open-skull approach. “We did not see plaques growing the way they do. They saw plaques getting five to six times bigger. We never saw that.”

Lee believes the different methods of preparing the cranial window may account for the apparent discrepancy. Though the thinned-skull method can cause damage to underlying brain tissue if not done carefully, his team chose to proceed with this approach after seeing extensive microglial and astrocytic activation in open-window preparations they had analyzed as a comparison. Other studies have also shown increased gliosis under open-skull but not thinned-skull cranial windows (see, e.g., Holtmaat et al., 2009 and Xu et al., 2007).

In contrast to the substantial plaque growth observed beneath the thin windows, Lee’s team saw very little growth through the open cranial windows they had prepared, reproducing what Hyman’s team had reported using the latter method (Meyer-Luehmann et al., 2008). “The plaques that pop up tend to be small, and they stay small,” Lee said.

In a separate set of experiments, Lee and colleagues treated six-month-old APP/PS1 mice with a γ-secretase inhibitor to determine whether reduced Aβ production affects plaque growth. Many in the field have assumed that plaques grow more slowly when the supply of soluble Aβ in the extracellular space dwindles, but few have attempted to observe directly whether this is true. Lee’s team did so by performing in-vivo microdialysis experiments in collaboration with coauthor David Holtzman, also at Washington University, to measure Aβ levels in the interstitial fluid (ISF) before and after treating mice with the γ-secretase inhibitor.

“If you decrease ISF Aβ by just a little bit, 25 percent over 24 hours, you can actually arrest plaque growth,” Lee said. These findings show “that γ-secretase modulators, or low-dose γ-secretase inhibitors, might be enough to lead to beneficial effects in patients,” Bacskai said. The researchers saw no such effect in 10-month-old mice, though, bolstering a growing view that treatment with Aβ-reducing compounds should begin before extensive plaque formation.

Bacskai remains at odds with the different conclusions on dynamics of plaque growth from the two studies. “Thin skulls are much harder to do, and limit what you can accomplish. You don't get to see as much of the brain,” Bacskai told ARF, noting that open-skull windows offer, in comparison, “a lot of real estate.” In addition, the open-skull approach allows repeat imaging of the same plaques over and over again, whereas with thin-skull windows, researchers can look at the same spot at most two or three times.

The open-skull method has drawbacks, too. “You’re taking out a part of the skull,” Lee said. “In effect, you’re introducing a foreign body—the glass coverslip. It's pretty invasive.”

Bacskai agreed that each technique has pros and cons. Even careful open-skull preparations may show minor damage at the edge of the window, he said, “but we always looked near the center.” Furthermore, switching from one method to another “is not like changing the pH of a buffer. It’s a surgical technique that you need to get good at, and it’s experience-dependent,” he said. “We don’t see this kind of gliosis at all if [the open-skull windows] are done correctly.” A recent study comparing the two methods (Holtmaat et al., 2009) suggested that the increased gliosis in open-skull windows did not seem to make a difference—at least for imaging synaptic spines. Bacskai and members of the Hyman lab have pioneered these in-vivo microscopy techniques for AD research, and Bacskai won a neuroimaging award for a separate study using these techniques at the International Conference on Alzheimer's Disease (ICAD) held last July in Vienna, Austria.

Nevertheless, the new data have made some scientists believe that choice of technique does matter. “This is excellent work,” Bart De Strooper, K.U. Leuven, Belgium, wrote in an e-mail to ARF. “The authors make elegantly the case that the procedures used to visualize amyloid plaques in vivo may strongly affect the generation and dynamics of the plaques.” Based on the new data, Gunnar Gouras, Weill Medical College of Cornell University, New York, suggested that the techniques could have distinct effects on nearby neurons. The findings “provide further evidence for the importance of inflammatory cells in modulating plaque pathology,” he wrote. “One wonders whether the different methods have a differential effect on neuritic dystrophy.” (See full comments below.)

Technical differences aside, Samir Kumar-Singh of the University of Antwerp, Belgium, thinks the new study’s conclusions about how long plaques take to grow “make intuitively more sense.” However, it “doesn’t really matter if it’s one day, two days, 10 days, or 15 days,” he said in a phone interview. “The idea is that plaques grow very quickly at the early stages,” and Aβ-reducing treatments should begin early in the pathogenic process. In an e-mail to ARF, Kumar-Singh noted that the dye (methoxy-X04) used in multiphoton microscopy “only binds fibrillar Aβ, not soluble/oligomeric forms of Aβ that most likely provide the initial nidus of plaque formation.” Therefore, he writes, “I don’t believe that we have had the final word on the temporal kinetics and dynamics of plaque formation.” (See full comment below.)

In the meantime, Lee and Bacskai are pursuing the question from a different angle—by determining what makes plaque growth grind to a halt. “Regardless of the exact rate of plaque formation, they have to stop growing at some point, because your whole brain doesn't turn into one gigantic plaque,” Bacskai said. “If you look at AD brain tissue at any stage, average plaque size is the same. Amyloid burden increases because you have an increased number of plaques, not because they’re bigger. So I would propose there’s some active process that makes a plaque stop growing.” Reactive glial cells could be key players, according to new data from Lee’s lab suggesting that activated astrocytes can inhibit plaque growth. Lee presented some of those unpublished findings at ICAD in Vienna.—Esther Landhuis

Comments

  1. This is an excellent study by Lee and colleagues employing serial multiphoton microscopy (MPM) to provide more clues to the process of plaque formation in the living brain of a well-established APP/PS1 transgenic mouse model of Alzheimer disease. Previous work by Hyman and colleagues had provided novel observations on the remarkably rapid appearance of plaques, and had also noted that once formed, there was little additional growth in the size of plaques. The focus of the current study is less the appearance and more the growth in the size of existing plaques over a time frame of a few weeks using a thinned skull window approach. They provide intriguing evidence for the importance of the type of window used to visualize plaques. Specifically, Lee and colleagues show that the open craniotomy with coverslip approach used in previous MPM studies in AD prevents the further growth of plaques and even augments regression of some plaques when compared with the thin-skull method. With the open- but not thin-skull method, there is marked cortical activation of inflammatory cells below the cranial window. These data also provide further evidence for the importance of inflammatory cells in modulating plaque pathology. One wonders whether the different methods have a differential effect on neuritic dystrophy. Interestingly, they also show that in younger but not older mice, γ-secretase inhibition retards formation and growth of new plaques while not effecting existing plaques. They suggest that these data support the importance of early rather than later therapeutic intervention in AD, although one can note that AD is also an anatomically progressive disease; less vulnerable brain regions may be at an earlier pathological stage (and therefore more amenable to treatment) than more vulnerable/pathologically advanced brain regions. Additionally, they show that the growth of plaques correlates with extracellular β amyloid levels in the interstitial fluid, a pool of β amyloid that many but not all view as the origin of plaques. Overall, this new MPM study is another important contribution in elucidating the development of β amyloid plaque pathology.

    View all comments by Gunnar Gouras

  2. Yan and colleagues add another piece to the plaque kinetics puzzle by showing, with on multiphoton in vivo microscopy, that amyloid plaques in a bigenic PSAPP mouse model appear and grow over a period of weeks before reaching a mature size. These data seem to be in apparent conflict with earlier work using the same technique on related mouse models (Meyer-Luehmann et al. 2008), where dense plaques were shown to reach their maximum size in about a day and thereafter maintain a status quo.

    The present study also goes forward to propose a reason for this discrepancy. Amyloid imaging through large open-skull cranial windows (as utilized solely by Meyer-Luehmann and colleagues) seems to activate gliosis, in contrast to thinned-skull windows of ≈1/10th the size, where calvaria are merely thinned down to allow in vivo microscopy without exposing the dura mater. This seems logical, as activation of gliosis has been shown in several studies to be an important factor in limiting plaque growth (Meyer-Luehmann et al. 2008; Bolmont et al., 2008; Yan et al., 2009). The stage of disease also seems to be important, as six-month-old mice with a higher proportion of smaller plaques demonstrate more accelerated plaque growth compared to 12-month-old animals (Yan et al., 2009).

    Secondly, however carefully studies attempt to show that the sizes of the plaques estimated by in vivo imaging are true representatives of the plaques occurring at that or a later stage of disease, it is always difficult to do so. Lastly, it’s important to keep in mind that the methoxy-X04 used in multiphoton in vivo microscopy only binds to fibrillar Aβ and not to the soluble/oligomeric forms of Aβ that most likely provide the initial nidus of plaque formation. For this reason I don’t believe that we have had the final word on the kinetics and dynamics of plaque formation. Important from a therapy point of view is that anti-Aβ treatments have to be started as early as possible in order to be efficacious—that everyone agrees on.

    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. PubMed.

    Bolmont T, Haiss F, Eicke D, Radde R, Mathis CA, Klunk WE, Kohsaka S, Jucker M, Calhoun ME. Dynamics of the microglial/amyloid interaction indicate a role in plaque maintenance. J Neurosci. 2008 Apr 16;28(16):4283-92. PubMed.

    View all comments by Samir Kumar-Singh

  3. This is excellent work. The authors make elegantly the case that the procedures used to visualize amyloid plaques in vivo may strongly affect the generation and dynamics of the plaques. It is also of strong interest that interstitial Aβ peptide is such an important contributor to the plaque dynamics, as this is a rather small pool of total Aβ in the brain, and also highly dynamic and influenced by medication. Finally, the fact that 20-30 percent changes in that pool strongly affect the plaque formation should indeed raise hope that a therapeutic window exists for secretase inhibitors.

    I strongly recommend the paper.

    View all comments by Bart De Strooper

  4. In my opinion, the discussion above misses one important fact: Brad Hyman's group published already in 2001 that plaques do not grow over time and that there is a restriction on plaque growth (Christie et al., 2001). In that study, more than 300 plaques were analyzed with two-photon microscopy over a time period of up to five months, and the investigators found the majority of plaques remained unchanged in size over time. Even more importantly, the data were observed using the thinned-skull method, i.e., the same method used by Yan et al., 2009. Therefore, thinned-skull versus open-skull preparation alone cannot account for the opposing result.

    References:

    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. PubMed.

    View all comments by Melanie Meyer-Luehmann

  5. We appreciate the comments of Dr. Meyer-Luehmann. However, the absence of plaque growth reported in the Christie et al. (2001) paper is very consistent with the data reported in our recent paper (Yan et al, 2009). Although we observed marked plaque growth in six-month-old APP/PS1 mice (early in plaque pathogenesis), we saw little to no growth in 10-month-old APP/PS1 mice. Of note, the Christie et al. paper did not see plaque growth in 18-month-old (mean age) Tg2576 mice. Therefore, our observations in older animals who have more advanced pathology are in agreement with the Christie et al. paper.

    References:

    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. PubMed.

    View all comments by Jin-Moo Lee

  6. This and other research demonstrates the deposition of Aβ in vivo in an animal model. Do we know that the “structures” that are shown being formed are also the same structures that are identified histo- or immunochemically postmortem?

    Thus, are we confident that what is observed is the full process that results in the structures that we identify classically as plaques postmortem?

    The alternative is that we are observing one part of a process. In some instances what is deposited may eventually be removed or transformed to something else and it is this “something else” which we identify postmortem as senile plaques.

    Are senile (neuritic) plaques simply deposits of Aβ, or are they more than this?

    View all comments by Chris Exley

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References

News Citations

  1. Popcorn Plaque? Alzheimer Disease Is Slow, Yet Plaque Growth Is Fast

Paper Citations

  1. 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. PubMed.
  2. Holtmaat A, Bonhoeffer T, Chow DK, Chuckowree J, De Paola V, Hofer SB, Hübener M, Keck T, Knott G, Lee WC, Mostany R, Mrsic-Flogel TD, Nedivi E, Portera-Cailliau C, Svoboda K, Trachtenberg JT, Wilbrecht L. Long-term, high-resolution imaging in the mouse neocortex through a chronic cranial window. Nat Protoc. 2009;4(8):1128-44. PubMed.
  3. Xu HT, Pan F, Yang G, Gan WB. Choice of cranial window type for in vivo imaging affects dendritic spine turnover in the cortex. Nat Neurosci. 2007 May;10(5):549-51. PubMed.

External Citations

  1. neuroimaging award

Further Reading

Papers

  1. 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. PubMed.
  2. Holtmaat A, Bonhoeffer T, Chow DK, Chuckowree J, De Paola V, Hofer SB, Hübener M, Keck T, Knott G, Lee WC, Mostany R, Mrsic-Flogel TD, Nedivi E, Portera-Cailliau C, Svoboda K, Trachtenberg JT, Wilbrecht L. Long-term, high-resolution imaging in the mouse neocortex through a chronic cranial window. Nat Protoc. 2009;4(8):1128-44. PubMed.
  3. Xu HT, Pan F, Yang G, Gan WB. Choice of cranial window type for in vivo imaging affects dendritic spine turnover in the cortex. Nat Neurosci. 2007 May;10(5):549-51. PubMed.

Primary Papers

  1. Yan P, Bero AW, Cirrito JR, Xiao Q, Hu X, Wang Y, Gonzales E, Holtzman DM, Lee JM. Characterizing the appearance and growth of amyloid plaques in APP/PS1 mice. J Neurosci. 2009 Aug 26;29(34):10706-14. PubMed.

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