Death by Glia?—Chemokine Receptor Nudges Neuron Loss in AD Mice

Amid brewing debate over whether microglia help or harm in Alzheimer disease, a study last year concluded, astonishingly, that the brain’s resident phagocytes had no impact on growth or clearance of Aβ plaques in AD transgenic mice (Grathwohl et al., 2009 and ARF related news story). Now, data reported online March 21 in Nature Neuroscience offer “a new perspective on what role microglia could play if not maintaining Aβ levels,” senior author Jochen Herms, Ludwig-Maximilians University, Munich, Germany, wrote in an e-mail to ARF. “We suggest that the main effect they have in AD may be facilitating nerve cell death.” Using in vivo two-photon imaging, Herms and colleagues showed they could detect neuronal loss in triple-transgenic AD mice, and that knocking out the microglial chemokine receptor CX3CR1 prevents this neurodegeneration.

Scores of studies have made the case for microglial involvement in AD, but it remains controversial under which conditions these immune cells intensify or curb pathogenesis (see ARF related news story on El Khoury et al., 2007 and Fan et al., 2007; ARF related news story on Ryu et al., 2009 and Koenigsknecht-Talboo et al., 2008). Much of the evidence suggesting microglia are beneficial comes from observations that the activated phagocytes migrate toward and chew up Aβ plaques in the brains of AD transgenic mice. Once spurred into action, though, microglia can also become troublemakers, unleashing proinflammatory cytokines and chemokines that harm surrounding cells.

In the current study, first authors Martin Fuhrmann, Tobias Bittner, and colleagues homed in on the mischievous side of microglia. They used two-photon imaging to look in vivo at these cells and their interactions with neighboring neurons in the brains of coauthor Frank LaFerla’s 3xTg mice, which overexpress pathogenic forms of presenilin-1 (M146V), amyloid precursor protein (Swedish), and tau (P301L). To make the neurons and microglia light up under the microscope, the researchers crossed the 3xTg mice with two additional transgenic lines—one that expresses yellow fluorescent protein (YFP) in subsets of layer III and V cortical neurons (Feng et al., 2000), and another carrying a green fluorescent protein (GFP) knock-in at the CX3CR1 locus (Jung et al., 2000). Inserted at this position, GFP not only labels microglia and other myeloid cells, but also interferes with neuron-microglia crosstalk in the new quintuple-transgenic model (5xTg). CX3CR1 is found on microglia and intercepts signals from CX3CL1 (aka fractalkine), a cytokine-like molecule expressed by neurons.

Two-photon imaging of four- to six-month-old YFP-expressing 3xTg mice revealed neuronal loss over two to four weeks of observation. The neurodegeneration was moderate—1.8 percent of YFP-positive layer III neurons lost over 28 days—but the fact it was seen at all is news in 3xTg mice, which develop plaques and tangles, but until this study never had detectable neuron loss. “The data are quite striking,” said Terrence Town, University of California, Los Angeles, who was not involved in the work. “The time-lapse images very clearly show that neurons are dying in the 3xTg mice, but in the fractalkine receptor knockout 3xTg mice, the neurons are persisting.” Moreover, in 3xTg mice, microglia swarmed in and moved more quickly around neurons that were about to be eliminated, whereas in CX3CR1-deficient 3xTg animals, microglia in similarly close proximity to neurons (i.e., within 100 micrometers) were comparatively fewer and slower. To Town, the data suggest that fractalkine/CX3CL1 signaling somehow calls microglia to neurons that are destined for death. “When you stop that pathway, you keep microglia from homing to those marked neurons,” he said.

What remains unclear, though, is whether microglia initiate the neuronal death cascade or simply serve as downstream mediators. Herms finds this a “most intriguing question,” but acknowledges it is hard to dissect. “In fact, we think that microglia do both—facilitate nerve cell death from a certain point of no return and subsequently remove the cell,” he wrote.

On a conceptual level (i.e., “Are microglia good or bad for AD?”), this idea departs from a recent report suggesting that microglia are irrelevant to formation and clearance of amyloid plaques in AD mice. In that study, co-led by Mathias Jucker, University of Tübingen, and Frank Heppner, Charite-Universitaetsmedizin, Berlin, researchers used a suicide gene approach to kill microglia for two to four weeks in two AD models (APP/PS1 and APP23). They found no change in plaque size or number (Grathwohl et al., 2009 and ARF related news story).

Considered alongside his own findings, Herms sees the two studies as complementary, even though they address different aspects of microglial involvement in AD. “If microglia have no role in plaque clearance, as suggested by Grathwohl et al., our study gives a hint at what their role might be instead—assisting neuron loss in AD,” he proposed. Furthermore, Herms and colleagues did measure insoluble Aβ40 levels in four- to six-month-old CX3CR1-deficient 3xTg mice and found no appreciable difference compared with 3xTg. This could be interpreted as saying the CX3CR1 receptor does not play a role in Aβ clearance or maintenance—or, more speculatively, that microglia have no part in these processes, Herms suggested.

A possible sticking point with the Aβ analyses is the fact that they were done in mice lacking hyperphosphorylated tau or extracellular amyloid plaques. At that age (four to six months), it is too early to determine whether CX3CR1 deficiency affects microglial control of amyloid deposition, noted Pritam Das of Mayo Clinic, Jacksonville, Florida, in an e-mail to ARF. Herms said his team is analyzing plaque load with respect to CX3CR1 loss in aged 5xTg mice but cannot comment on this issue at present.

Meanwhile, CX3CR1’s role in AD is turning out to be a complex story. At a Keystone symposium called Alzheimer’s Disease Beyond Aβ, held 10-15 January 2010 at Copper Mountain, Colorado, parallel studies led by Joseph El Khoury of Harvard Medical School and by Richard Ransohoff and Bruce Lamb of the Cleveland Clinic, Ohio, showed that loss of CX3CR1 led to reduced amyloid deposition in APP/PS1 mice. In another study from Lamb’s lab, CX3CR1 knockouts crossed with humanized tau (hTau) transgenic mice showed worse tau pathology compared with CX3CR1-sufficient hTau mice (see ARF related conference story). “It appears that the altered microglial reaction in CX3CR1 knockout mice is a double-edged sword, producing better amyloid phagocytosis and worse tau pathology,” Ransohoff wrote in an e-mail to ARF (see full comment below and Ransohoff, 2009).

One aspect of the microglial effects in the APP/PS1 studies—namely, migration—did seem to jibe with Herms’s data, Town noted. El Khoury reported at Keystone that CX3CR1 knockout APP/PS1 mice had fewer mononuclear phagocytes associating with Aβ plaques. In Herms’s study, lack of CX3CR1 also slowed migration of phagocytes—but toward neurons.—Esther Landhuis

Comments

  1. In two parallel, separate studies, Joe El Khoury and we (a group led by Bruce Lamb and including Sungho Lee, Nick Varvel, and myself) crossed CX3CR1 KOs to APP-PS1 mice (using distinct APP-PS1 models, ours from Matthias Jucker; El Khoury’s from Dave Borchelt) and monitored amyloid deposition. Our results were entirely concordant (using slightly different methods of analysis): there was a strong, gene dosage-dependent decrease in amyloid deposition in the CX3CR1 KO mice. This decrease was not associated with evident change in APP expression, nor in processing. Further, there were fewer microglia associated with each core plaque in the CX3CR1 KOs. The hypothesis was that CX3CR1 KO microglia are more efficient at amyloid phagocytosis, therefore clearing more with fewer cells. Since then, Bruce’s lab has in vitro data to support this hypothesis. These findings (obtained independently by our lab and that of El Khoury) are neither concordant nor discordant with those from Herms et al: their assessment of insoluble Aβ appears to show a non-significant reduction in the KO mice (Fig 1I), although the small number of animals assessed might preclude statistical significance.

    In another study from Bruce’s group (Kiran Bhaskar), CX3CR1 KO mice (crossed with mice ‘humanized’ for tau [hTau mice]) showed worse tau pathology, dependent on IL1 and p38MAP kinase and resulting in cognitive impairment.

    In response, then, to the key question about microglia in general and CX3CR1 in particular, it appears that the altered microglial reaction in CX3CR1 KO mice is a double-edged sword, producing better amyloid phagocytosis and worse tau pathology.

    Combining the two aspects of AD pathology (in the triple-Tg) and focusing on a novel assay for neuron loss (monitoring with two-photon imaging), Herms et al. showed benefit related to absence of CX3CR1. Their work represents (to our knowledge) the first evidence for neuron loss in the triple-Tg AD model, and one which would not be observed using stereology (1.8 percent of neurons within one month). It remains uncertain why a uniform 1.8 percent neuron loss would not, however, be recognized if it persisted for six to 12 months. The authors’ hypothesis that microglial activation precedes neuron loss and therefore is causative needs further study: injury to neurons activates microglia, and it can clearly be seen in Fig. 1 (compare day 0 in 1c and 1e) that the +/- microglia are already activated. This conclusion becomes even more solid when one considers that the -/- microglia have two copies of GFP, while the +/- microglia have one and, if imaged similarly, would appear smaller and less prominent.

    In summary, Herms et al. have shown neuronal cell loss in an AD model using two-photon imaging, and have provided evidence that microglial CX3CR1 is involved, somehow, in that process. The relationship to amyloid deposition or toxicity, or to tau pathology, needs to be studied further at the mechanistic level.

    View all comments by Richard Ransohoff

  2. The recent report from the Herms group offers new insight into the enigmatic relationship between microglia and AD pathobiology. The authors have focused on whether fractalkine receptor on microglial cells participates in neuronal loss using Frank LaFerla’s 3xTg-AD model. The novelty in this paper is really twofold: demonstration of in vivo neuronal loss in real-time, and new biology showing the role of microglial fractalkine receptor (CX3CR1) in mediating this neuronal death.
    The authors should be commended for taking such an elegant approach, utilizing two-photon intravital imaging. It is interesting that these authors observe neuronal loss within two weeks in fractalkine receptor-sufficient 3xTg-AD mice. This report comes on the heels of another recent Nature Neuroscience paper from Mathias Jücker’s group, where those authors used a ganciclovir cd11b suicide gene approach to destroy microglia in a transgenic APP/PS1 mouse model of AD for two to four weeks. Surprisingly, those authors did not detect altered cerebral amyloidosis or amyloid-associated neuritic dystrophy in AD model mice that were microglia-deficient. When taking the Jücker report together with this present work, one wonders whether there are not AD mouse model-specific effects of microglia. Of course, the only way to answer such a question would be to reproduce both sets of findings in other AD animal models.

    I’d like to comment on the present authors’ data showing that fractalkine receptor-sufficient microglia increase in velocity when moving toward the neurons that are marked for death prior to the actual neuronal loss. Perhaps one of the more penetrating questions is, Are microglia initiating neuronal loss or acting at a point downstream, but still on the pathway to, neuronal death? I am sure that we will continue to grapple with this and other questions that have been prompted by this interesting work.

    View all comments by Terrence Town

  3. This paper by Fuhrmann et al. shows elegant two-photon imaging of neurons and microglia in the 3x transgenic model. It is certainly a technical tour de force. The most striking result is that there is neuron loss in this model, which has not been previously described. The numbers are low, however, tens of neurons per cubic millimeter of cortex per month, which is probably much less than 1 percent of the total (YFP-positive neurons are only a subset of neurons). Is this too few to come to a definitive conclusion about the role of CX3CR1 in neurodegeneration? Perhaps, but the best way to address this would be a more low-tech approach in a model with significant neuronal loss. Nonetheless, addressing the role of microglia in the AD brain is important, and these results are certainly intriguing.

    View all comments by Brian Bacskai

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References

News Citations

  1. The Brain Minus Microglia—No Effect on Plaques
  2. Microglia—Medics or Meddlers in Dementia
  3. Fatal Attraction or Beneficial Interaction: Microglia, Aβ and Neurons
  4. Copper Mountain: Fractious Receptors, Glia, and AD Pathology

Paper Citations

  1. Grathwohl SA, Kälin RE, Bolmont T, Prokop S, Winkelmann G, Kaeser SA, Odenthal J, Radde R, Eldh T, Gandy S, Aguzzi A, Staufenbiel M, Mathews PM, Wolburg H, Heppner FL, Jucker M. Formation and maintenance of Alzheimer's disease beta-amyloid plaques in the absence of microglia. Nat Neurosci. 2009 Nov;12(11):1361-3. PubMed.
  2. 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. PubMed.
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  5. Koenigsknecht-Talboo J, Meyer-Luehmann M, Parsadanian M, Garcia-Alloza M, Finn MB, Hyman BT, Bacskai BJ, Holtzman DM. Rapid microglial response around amyloid pathology after systemic anti-Abeta antibody administration in PDAPP mice. J Neurosci. 2008 Dec 24;28(52):14156-64. PubMed.
  6. Feng G, Mellor RH, Bernstein M, Keller-Peck C, Nguyen QT, Wallace M, Nerbonne JM, Lichtman JW, Sanes JR. Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP. Neuron. 2000 Oct;28(1):41-51. PubMed.
  7. Jung S, Aliberti J, Graemmel P, Sunshine MJ, Kreutzberg GW, Sher A, Littman DR. Analysis of fractalkine receptor CX(3)CR1 function by targeted deletion and green fluorescent protein reporter gene insertion. Mol Cell Biol. 2000 Jun;20(11):4106-14. PubMed.
  8. Ransohoff RM. Chemokines and chemokine receptors: standing at the crossroads of immunobiology and neurobiology. Immunity. 2009 Nov 20;31(5):711-21. PubMed.

Other Citations

  1. 3xTg mice

Further Reading

Papers

  1. Ransohoff RM. Chemokines and chemokine receptors: standing at the crossroads of immunobiology and neurobiology. Immunity. 2009 Nov 20;31(5):711-21. PubMed.
  2. Grathwohl SA, Kälin RE, Bolmont T, Prokop S, Winkelmann G, Kaeser SA, Odenthal J, Radde R, Eldh T, Gandy S, Aguzzi A, Staufenbiel M, Mathews PM, Wolburg H, Heppner FL, Jucker M. Formation and maintenance of Alzheimer's disease beta-amyloid plaques in the absence of microglia. Nat Neurosci. 2009 Nov;12(11):1361-3. PubMed.
  3. Hanisch UK, Kettenmann H. Microglia: active sensor and versatile effector cells in the normal and pathologic brain. Nat Neurosci. 2007 Nov;10(11):1387-94. PubMed.
  4. Wyss-Coray T. Inflammation in Alzheimer disease: driving force, bystander or beneficial response?. Nat Med. 2006 Sep;12(9):1005-15. PubMed.

Primary Papers

  1. Fuhrmann M, Bittner T, Jung CK, Burgold S, Page RM, Mitteregger G, Haass C, Laferla FM, Kretzschmar H, Herms J. Microglial Cx3cr1 knockout prevents neuron loss in a mouse model of Alzheimer's disease. Nat Neurosci. 2010 Apr;13(4):411-3. PubMed.

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