The demonstration by Grathwohl et al. that substantial depletion of microglia has no consequences for Aβ deposition is indeed intriguing. However, this finding must be taken in context of a good deal of data indicating that microglia do participate in the sequelae of events occurring in AD and in APP transgenic models, much of which come from studies enlisting elegant gene- or cell-ablation approaches such as those applied here. For instance, genetic ablation of Toll-like receptor 2 (Richard et al., 2008) or CCR2 (El Khoury et al., 2007) exacerbates plaque deposition.

More importantly, many hypotheses about the roles of microglia in AD involve events downstream of amyloidogenesis, such as synaptic dysregulation or frank neurotoxicity. The sole parameter assessed in this paper that has any possible link to such downstream events was APP staining in dystrophic neurites. But no compelling claims had ever been made for a connection between microglial actions and these structures; the APP staining is likely a consequence of the transgene itself. It would be more relevant to...  Read more

The demonstration by Grathwohl et al. that substantial depletion of microglia has no consequences for Aβ deposition is indeed intriguing. However, this finding must be taken in context of a good deal of data indicating that microglia do participate in the sequelae of events occurring in AD and in APP transgenic models, much of which come from studies enlisting elegant gene- or cell-ablation approaches such as those applied here. For instance, genetic ablation of Toll-like receptor 2 (Richard et al., 2008) or CCR2 (El Khoury et al., 2007) exacerbates plaque deposition.

More importantly, many hypotheses about the roles of microglia in AD involve events downstream of amyloidogenesis, such as synaptic dysregulation or frank neurotoxicity. The sole parameter assessed in this paper that has any possible link to such downstream events was APP staining in dystrophic neurites. But no compelling claims had ever been made for a connection between microglial actions and these structures; the APP staining is likely a consequence of the transgene itself. It would be more relevant to survey markers of synapse integrity and function, as well as tau phosphorylation. Indeed, several lines of investigation indicate that microglia play tonic roles in normal synapse formation, modulation, and removal (Bessis et al., 2007; Wake et al., 2009). If this is the case, it is difficult to imagine that their activation in AD does not impact synaptic function in some way.

There is also compelling evidence that one consequence of FAD mutations is dependent upon microglia; namely, microglia expressing mutant forms of presenilin-1 alter the fate of neural progenitor cells (Choi et al., 2008). Relatedly, it would certainly be important to characterize the behavioral profile of mice manipulated in the manner of Grathwohl et al.

References:
Bessis A, Béchade C, Bernard D, Roumier A. Microglial control of neuronal death and synaptic properties. Glia. 2007 Feb;55(3):233-8. Abstract

Choi SH, Veeraraghavalu K, Lazarov O, Marler S, Ransohoff RM, Ramirez JM, Sisodia SS. Non-cell-autonomous effects of presenilin 1 variants on enrichment-mediated hippocampal progenitor cell proliferation and differentiation. Neuron. 2008 Aug 28;59(4):568-80. 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 13:432–438. Abstract

Richard KL, Filali M, Préfontaine P, Rivest S. Toll-like receptor 2 acts as a natural innate immune receptor to clear amyloid beta 1-42 and delay the cognitive decline in a mouse model of Alzheimer's disease. J. Neurosci. 2008 May 28;28(22):5784-93. Abstract

Wake H, Moorhouse AJ, Jinno S, Kohsaka S, Nabekura J. Resting microglia directly monitor the functional state of synapses in vivo and determine the fate of ischemic terminals. J Neurosci. 2009 Apr 1;29(13):3974-80. Abstract

View all comments by Steve Barger

I would like to thank Erene Mina and Drs. Walker and Jucker. They provide insightful comments regarding specific aspects of the study. I'd like to address a few points here.

The first one regards irradiation and its effects on the blood-brain barrier (BBB). There is not very strong evidence that irradiation alters the BBB, and brain infiltration of bone marrow-derived cells has been reported with other techniques as well. Messengale and colleagues have validated this concept using both lethal irradiation and parabiosis techniques in mice (Massengale et al., 2005). Although most (if not all) GFP cells found in the brains of chimeric mice have a microglial phenotype, the overall contributions of such cells to the brain-resident microglial populations of normal mice remain quite low (e.g., 0.5-11.5 percent of resident microglia). This is what we generally observe in our mice (Simard and Rivest, 2004). In APP mice, however, there is a robust microglial recruitment toward the plaques, and those that derive from the bone marrow are attracted at a specific time of the disease....  Read more

I would like to thank Erene Mina and Drs. Walker and Jucker. They provide insightful comments regarding specific aspects of the study. I'd like to address a few points here.

The first one regards irradiation and its effects on the blood-brain barrier (BBB). There is not very strong evidence that irradiation alters the BBB, and brain infiltration of bone marrow-derived cells has been reported with other techniques as well. Messengale and colleagues have validated this concept using both lethal irradiation and parabiosis techniques in mice (Massengale et al., 2005). Although most (if not all) GFP cells found in the brains of chimeric mice have a microglial phenotype, the overall contributions of such cells to the brain-resident microglial populations of normal mice remain quite low (e.g., 0.5-11.5 percent of resident microglia). This is what we generally observe in our mice (Simard and Rivest, 2004). In APP mice, however, there is a robust microglial recruitment toward the plaques, and those that derive from the bone marrow are attracted at a specific time of the disease. Other groups have observed a similar pattern in irradiated APP mice (Malm et al., 2005; Stalder et al., 2005), and one can appreciate the robust microglia infiltration in the plaques of non-irradiated mice (Fig. 1 and supp. movie 1). Therefore, I do not think that infiltration is caused by alteration of the BBB in irradiated mice, but is a natural process that is especially dynamic while the plaques progress. The mechanisms explaining why bone marrow-derived microglia are no longer recruited toward the plaques at a specific time point in the disease have yet to be unraveled with future experiments.

Another point raised is that we did not look at the colocalization of Aβ in the lysosomes of the resident microglia. We actually did a meticulous analysis of such processes in the chimeric APP, and while GFP cells were almost always associated with lysosomal Aβ, the resident cells were not. This is the reason that we did not show these results, but we have discussed them.

We observed phagocytosis by bone marrow-derived microglia during a very specific time. This takes place around 6 months of age in the APP/PS1 mice, and we no longer see these cells at 9 months. Therefore, cell recruitment (of bone marrow origin) and phagocytosis are dynamic and transient phenomena, which may explain why other groups have not detected it. This also explains why inhibition of cell recruitment (APP/TK mice) from 5 to 6 months has such profound consequences on plaque growth. We are now working on new genetic strategies to enhance and improve the recruitment of these cells for a longer period of time to see if we can prevent the amyloid cascade and cognitive deficit.

Finally, multiple staining and 3D reconstructions using confocal laser-scanning microscopy are powerful tools to determine cellular compartmentalization, such as Aβ within the lysosomal GFP cells.

References:
Malm TM, Koistinaho M, Parepalo M, Vatanen T, Ooka A, Karlsson S, Koistinaho J. Bone-marrow-derived cells contribute to the recruitment of microglial cells in response to β-amyloid deposition in APP/PS1 double transgenic Alzheimer mice. Neurobiol Dis. 2005 Feb;18(1):134-42. Abstract

Massengale M, Wagers AJ, Vogel H, Weissman IL. Hematopoietic cells maintain hematopoietic fates upon entering the brain. J Exp Med. 2005 May 16;201(10):1579-89. Abstract

Simard AR, Rivest S. Bone marrow stem cells have the ability to populate the entire central nervous system into fully differentiated parenchymal microglia. FASEB J. 2004 Jun;18(9):998-1000. Epub 2004 Apr 14. 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

Stalder AK, Ermini F, Bondolfi L, Krenger W, Burbach GJ, Deller T, Coomaraswamy J, Staufenbiel M, Landmann R, Jucker M. Invasion of hematopoietic cells into the brain of amyloid precursor protein transgenic mice. J Neurosci. 2005 Nov 30;25(48):11125-32. Abstract

View all comments by Serge Rivest

The paper from Joseph El Khoury and colleagues presents convincing evidence that the absence of activated microglia is detrimental in the Tg2576 model. On the surface, from our study in J. Neuroscience, one may conclude that microglial activation is harmful. It likely depends on the context of how you are viewing the problem. Early on, microglial activation may be helpful by facilitating clearance of Aβ from brain; in their absence more Aβ accumulates (El Khoury). On the other hand, if Aβ is not cleared and microglia remain activated, this may lead to the chronic neuroinflammation and behavioral deficits that we observed in our model.

Another caveat that we must all recognize is what are the specific features of the models we work with. Each has its own strengths and weaknesses for studying specific aspects of Aβ pathology. For example, the widely used Tg2576 mouse expresses high amounts of Swedish mutant human APP in many cell types, producing high amounts of wild-type Aβ peptides and parenchymal amyloid plaques. The Tg-SwDI mouse expresses low...  Read more

The paper from Joseph El Khoury and colleagues presents convincing evidence that the absence of activated microglia is detrimental in the Tg2576 model. On the surface, from our study in J. Neuroscience, one may conclude that microglial activation is harmful. It likely depends on the context of how you are viewing the problem. Early on, microglial activation may be helpful by facilitating clearance of Aβ from brain; in their absence more Aβ accumulates (El Khoury). On the other hand, if Aβ is not cleared and microglia remain activated, this may lead to the chronic neuroinflammation and behavioral deficits that we observed in our model.

Another caveat that we must all recognize is what are the specific features of the models we work with. Each has its own strengths and weaknesses for studying specific aspects of Aβ pathology. For example, the widely used Tg2576 mouse expresses high amounts of Swedish mutant human APP in many cell types, producing high amounts of wild-type Aβ peptides and parenchymal amyloid plaques. The Tg-SwDI mouse expresses low levels of Swedish/Dutch/Iowa mutant human APP only in neurons producing low levels of vasculotropic Dutch/Iowa mutant Aβ peptides and microvascular amyloid deposits. In light of these differences in the models, some variations in results may be attributed to the sites of amyloid deposition and possibly due to differences in microglial responses to wild-type and vasculotropic mutant Aβ peptides and amyloid deposits.

View all comments by William Van Nostrand

El Khoury et al. have produced a dataset that adds to those indicating a beneficial role for monocytic phagocytes (either activated microglia or hematogenous macrophages) with respect to the development of Alzheimer-related pathology. Some data have indicated that inflammation-related events elaborated by microglia contribute to AD pathology. This includes the overexpression of interleukin-1-β in APP-transgenic mouse models of AD, as well as attenuation of Aβ accumulation in these mice by anti-inflammatory agents such as ibuprofen and, more recently, minocycline (see Fan et al., 2007). But beginning with paradigms in which such mice are immunized against Aβ, increasing evidence has suggested that monocyte-derived cells can help to clear Aβ from the brain through phagocytosis and/or expression of Aβ-degrading proteases. For instance, Morgan and colleagues have shown that injection of the powerful inflammatory agent lipopolysaccharide into APP-transgenic mice results in Aβ clearance (DiCarlo et al., 2006), and the clearance or prevention of Aβ...  Read more

El Khoury et al. have produced a dataset that adds to those indicating a beneficial role for monocytic phagocytes (either activated microglia or hematogenous macrophages) with respect to the development of Alzheimer-related pathology. Some data have indicated that inflammation-related events elaborated by microglia contribute to AD pathology. This includes the overexpression of interleukin-1-β in APP-transgenic mouse models of AD, as well as attenuation of Aβ accumulation in these mice by anti-inflammatory agents such as ibuprofen and, more recently, minocycline (see Fan et al., 2007). But beginning with paradigms in which such mice are immunized against Aβ, increasing evidence has suggested that monocyte-derived cells can help to clear Aβ from the brain through phagocytosis and/or expression of Aβ-degrading proteases. For instance, Morgan and colleagues have shown that injection of the powerful inflammatory agent lipopolysaccharide into APP-transgenic mice results in Aβ clearance (DiCarlo et al., 2006), and the clearance or prevention of Aβ deposits in immunized mice is associated with some signs that microglia are more active.

An important question has been whether these beneficial roles of monocytic phagocytes are operative in the basal condition (and eventually overwhelmed in the development of disease) or are instead induced only by extraordinary manipulation, such as immunization or injections of lipopolysaccharide. El Khoury’s approach was to remove or reduce a chemokine receptor (CCR2) responsible for trafficking microglia and/or peripheral macrophages to sites of inflammation, which would include amyloid plaques in the APP transgenic mice. The resulting increase in Aβ accumulation (both soluble and deposits), coupled with an absence of the accumulation of monocytic phagocytes that normally arises in APP transgenics, suggests that monocyte-derived cells tonically participate in the removal of Aβ; microglia from the CCR2-knockout mice still reacted to Aβ in culture. This specific requirement for chemotaxis, then, is consistent with recent studies showing the homing of bone marrow-derived monocytic cells to plaques in APP transgenics (Simard et al., 2006). Microglia are so extensively distributed throughout the cortex that one should imagine they scarcely need to migrate if they were the primary mediators of Aβ clearance.

Of course, the caveat that an APP transgenic mouse is not a human with AD goes without saying. And that may be most relevant to the interpretation of what happens downstream of Aβ clearance. El Khoury et al. reported a decrease in lifespan in the CCR2-knockout animals, but this may have been due to cerebrovascular hemorrhage. It is possible that well-intentioned clearance of Aβ, regardless of how successful, may produce byproducts that interfere with neurophysiology. To wit, the application of the anti-inflammatory antibiotic minocycline by Fan et al. protected against behavioral deficits in APP transgenic mice without altering Aβ levels or deposition, and ibuprofen treatment is associated with a decrease in a marker of apoptosis per plaque rather than a reduction in plaques themselves (Lim et al., 2001). Thus, strategies aimed at optimizing the impact of inflammatory processes or monocytic phagocytes on AD pathogenesis should take into account the potential requirement of a balance between the benefits of Aβ clearance and the maladaptive consequences of inflammatory sequelae on neuronal function and viability.

It is somewhat unfortunate that El Khoury et al. utilized an APP-transgenic strain that has a mixed genetic background (SJL x C57BL/6). Aβ deposition is notoriously strain-dependent, with the relevant alleles remaining unknown. Any cross of a mixed background creates the opportunity for genetic variability in the progeny, even in littermates. This concern can be mitigated by analyzing sufficient numbers. El Khoury et al. used as few as three or four animals per group, which seems low except for the fact that techniques were applied which precluded the use of the same animals for some of the techniques (e.g., immunohistochemistry vs. FACS); thus, the true numbers of animals over which dramatic differences were seen was actually six or seven per genotype.

References:
Fan R, Xu F, Previti ML, Davis J, Grande AM, Robinson JK, Van Nostrand WE. Minocycline reduces microglial activation and improves behavioral deficits in a transgenic model of cerebral microvascular amyloid. J Neurosci. 2007 Mar 21;27(12):3057-63. Abstract

DiCarlo G, Wilcock D, Henderson D, Gordon M, Morgan D. Intrahippocampal LPS injections reduce Aβ load in APP+PS1 transgenic mice. Neurobiol Aging. 2001 Nov-Dec;22(6):1007-12. 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

Lim GP, Yang F, Chu T, Gahtan E, Ubeda O, Beech W, Overmier JB, Hsiao-Ashe K, Frautschy SA, Cole GM. Ibuprofen effects on Alzheimer pathology and open field activity in APPsw transgenic mice. Neurobiol Aging. 2001 Nov-Dec;22(6):983-91. Abstract

View all comments by Steve Barger

It is odd that an effect was noted by El Khoury et al. in a Ccr2 knockout. Cedric Raines showed in a landmark paper that Ccr2 was so redundant that it made no impact on trafficking of monocyte-related cells in EAE (experimental autoimmune encephalomyelitis).

References:
Gaupp S, Pitt D, Kuziel WA, Cannella B, Raine CS. Experimental autoimmune encephalomyelitis (EAE) in CCR2(-/-) mice: susceptibility in multiple strains. Am J Pathol. 2003;162:139-50. Abstract

View all comments by Bo Hu

The report by El Khoury and colleagues shows that recruitment of macrophage-like cells to the brains of Tg2576 mice via Ccr2 plays an important role in limiting AD-like pathology. This is a very interesting finding and extends the work of Stalder et al. (2005), who noted the presence of round, non-process-bearing, macrophage-like cells in APP23 mice with appreciable amyloid deposits.

El Khoury et al. have gone further by establishing that Ccr2-dependent recruitment of microglia/macrophage-like cells is important in limiting progression of cerebral amyloidosis. If taken to the logical endpoint, this would mean that microglia and/or macrophages serve to limit amyloidosis by phagocytosing/clearing amyloid deposits in AD mice in the absence of genetic manipulation (and perhaps something similar may occur in AD patients). However, careful 3D reconstruction of microglia and amyloid in APP23 or Tg2576 mice fails to show this (Stalder et al., 2001; Wegiel et al., 2004).

An alternate explanation is that microglia/macrophages secrete a soluble factor (e.g., a cytokine or...  Read more

The report by El Khoury and colleagues shows that recruitment of macrophage-like cells to the brains of Tg2576 mice via Ccr2 plays an important role in limiting AD-like pathology. This is a very interesting finding and extends the work of Stalder et al. (2005), who noted the presence of round, non-process-bearing, macrophage-like cells in APP23 mice with appreciable amyloid deposits.

El Khoury et al. have gone further by establishing that Ccr2-dependent recruitment of microglia/macrophage-like cells is important in limiting progression of cerebral amyloidosis. If taken to the logical endpoint, this would mean that microglia and/or macrophages serve to limit amyloidosis by phagocytosing/clearing amyloid deposits in AD mice in the absence of genetic manipulation (and perhaps something similar may occur in AD patients). However, careful 3D reconstruction of microglia and amyloid in APP23 or Tg2576 mice fails to show this (Stalder et al., 2001; Wegiel et al., 2004).

An alternate explanation is that microglia/macrophages secrete a soluble factor (e.g., a cytokine or protease) that limits cerebral amyloidosis. Yet, the converse—that reactive glia produce acute phase reactants/cytokines such as ApoE, ACT and IL-1 that promote amyloidosis—has been shown (Potter et al., 2001; Nilsson et al., 2001). In light of these reports, what is the authors’ take on the mechanism responsible for their finding?

El Khoury et al. also report that Ccr2 deletion limits the lifespan of Tg2576 animals, and suggest that there is a connection between increased AD-like pathology in Ccr2-deficient Tg2576 mice and their premature death. This conclusion should be taken with caution. Although not often pointed out, Tg2576 mice actually overexpress the mutant human APP transgene in regions other than the brain (for example, peripheral vascular smooth muscle cells and endothelial cells), and it is well-established that transgene-derived Aβ is easily detected systemically in these mice, so early death of Ccr2-deficient Tg2576 mice may be CNS-independent.

The paper by Fan and colleagues presents an interesting set of results that suggest dampening microglial activation via minocycline treatment is beneficial in their mouse model of vascular amyloidosis. Interestingly, they found reduction in “activated” microglia that corresponded with mitigation of behavioral impairment. Their results fit well with the work of Greg Cole’s group, who showed that treatment of Tg2576 mice with the non-steroidal anti-inflammatory drug (NSAID) ibuprofen or the naturally occurring NSAID curcumin reduces microglial activation concomitant with reduced cerebral amyloidosis and behavioral impairment (Lim et al., 2000; Lim et al, 2001a; Lim et al., 2001b). Fan and colleagues’ data also fit well with our previous results showing that genetic or pharmacologic interruption of CD40-CD40 ligand interaction mitigates microglial activation in response to Aβ peptides, and reduces microgliosis, cerebral amyloidosis, and behavioral impairment in AD mouse models (Tan, Town et al., 1999; Tan, Town et al., 2002; Town et al., 2001; Todd Roach et al., 2004).

When taken together, the studies suggest that “activation” of microglia/macrophages is not simply one phenotype. We have suggested that these innate immune cells may respond with a range of responses from pro-phagocytic/anti-inflammatory to anti-phagocytic/proinflammatory (Town et al., 2005). Understanding the molecular underpinnings of these various responses of microglia/macrophages will likely be key in targeting these cells for therapeutic intervention in neurodegenerative diseases (particularly AD).

References:
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. Nature Medicine. 2007, March 11. Advanced online publication. Abstract

Fan R, Xu F, Previti ML, Davis J, Grande AM, Robinson JK, Van Nostrand WE. Minocycline reduces microglial activation and improves behavioral deficits in a transgenic model of cerebral microvascular amyloid. J Neurosci. 2007;27(12):3057-63. Abstract

Lim GP, Yang F, Chu T, Chen P, Beech W, Teter B, Tran T, Ubeda O, Ashe KH, Frautschy SA, Cole GM. Ibuprofen suppresses plaque pathology and inflammation in a mouse model for Alzheimer's disease. Journal of Neuroscience. 2000 Aug 1;20(15):5709-14. Abstract

Lim GP, Yang F, Chu T, Gahtan E, Ubeda O, Beech W, Overmier JB, Hsiao-Ashec K, Frautschy SA, Cole GM. Ibuprofen effects on Alzheimer pathology and open field activity in APPsw transgenic mice. Neurobiology of Aging. 2001;22(6):983-91. Abstract

Lim GP, Chu T, Yang F, Beech W, Frautschy SA, Cole GM. The curry spice curcumin reduces oxidative damage and amyloid pathology in an Alzheimer transgenic mouse. Journal of Neuroscience. 2001;21(21):8370-7. Abstract

Nilsson LN, Bales KR, DiCarlo G, Gordon MN, Morgan D, Paul SM, Potter H. Alpha-1-antichymotrypsin promotes beta-sheet amyloid plaque deposition in a transgenic mouse model of Alzheimer's disease. Journal of Neuroscience. 2001;21(5):1444-51. Abstract

Potter H, Wefes IM, Nilsson LN. The inflammation-induced pathological chaperones ACT and apo-E are necessary catalysts of Alzheimer amyloid formation. Neurobiology of Aging. 2001;22(6):923-30. Abstract

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

Stalder AK, Ermini F, Bondolfi L, Krenger W, Burbach GJ, Deller T, Coomaraswamy J, Staufenbiel M, Landmann R, Jucker M. Invasion of hematopoietic cells into the brain of amyloid precursor protein transgenic mice. Journal of Neuroscience. 2005;25(48):11125-32. Abstract

Tan J, Town T, Paris D, Mori T, Suo Z, Crawford F, Mattson MP, Flavell RA, Mullan M. Microglial activation resulting from CD40-CD40L interaction after beta-amyloid stimulation. Science. 1999;286(5448):2352-5. Abstract

Tan J, Town T, Crawford F, Mori T, DelleDonne A, Crescentini R, Obregon D, Flavell RA, Mullan MJ. Role of CD40 ligand in amyloidosis in transgenic Alzheimer's mice. Nature Neuroscience. 2002;5(12):1288-93. Abstract

Todd Roach J, Volmar CH, Dwivedi S, Town T, Crescentini R, Crawford F, Tan J, Mullan M. Behavioral effects of CD40-CD40L pathway disruption in aged PSAPP mice. Brain Research. 2004;1015(1-2):161-8. Abstract

Town T, Tan J, Mullan M. CD40 signaling and Alzheimer's disease pathogenesis. Neurochemistry International. 2001;39(5-6):371-80. Abstract

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

Wegiel J, Imaki H, Wang KC, Wegiel J, Rubenstein R. Cells of monocyte/microglial lineage are involved in both microvessel amyloidosis and fibrillar plaque formation in APPsw tg mice. Brain Research. 2004;1022(1-2):19-29. Abstract

View all comments by Terrence Town

Meyer-Luehmann and colleagues provide new insights into the temporal sequence of events surrounding amyloid plaque formation and the brain’s cellular responses to this formation. It is exciting to see that the rapid formation of plaques that had been predicted by previously published reports using in vitro techniques (Vitek et al., 1994; Jarrett et al., 1993) actually occurs in vivo. The concept of seeding by submicroscopic Aβ particles clearly remains an important mechanism for plaque formation and deposition.

Useful insights are also provided by visualization of the microglial response to the newly formed amyloid plaques. Microglia accumulate at the plaques, indicating the presence of activating/migration signals, most likely from Aβ. This, plus the microglial morphological changes, suggest that a pre-programmed response pattern, which is typical of macrophages involved in the innate immune response, has been initiated. However, it is clear from the visual data that the term “microglia activation” needs to be reconsidered and redefined. Although functional changes...  Read more

Meyer-Luehmann and colleagues provide new insights into the temporal sequence of events surrounding amyloid plaque formation and the brain’s cellular responses to this formation. It is exciting to see that the rapid formation of plaques that had been predicted by previously published reports using in vitro techniques (Vitek et al., 1994; Jarrett et al., 1993) actually occurs in vivo. The concept of seeding by submicroscopic Aβ particles clearly remains an important mechanism for plaque formation and deposition.

Useful insights are also provided by visualization of the microglial response to the newly formed amyloid plaques. Microglia accumulate at the plaques, indicating the presence of activating/migration signals, most likely from Aβ. This, plus the microglial morphological changes, suggest that a pre-programmed response pattern, which is typical of macrophages involved in the innate immune response, has been initiated. However, it is clear from the visual data that the term “microglia activation” needs to be reconsidered and redefined. Although functional changes in the microglia were not measured, it is highly likely that a “proinflammatory” or classical activation sequence that is typically associated with severe tissue damage is limited either in time or amount. Other than the dystrophic neurites that could also be observed in non-plaque areas, there is no overt evidence of dying neurons in or around the plaques. While this lack of morphological evidence does not formally exclude the possibility that dying neurons are associated with plaques, the microglial response observed is consistent with what appears to be a “walling-off” response typical of an alternatively activated microglia.

Alternative activation is a response pattern of macrophages that is associated with fibrosis and tissue repair. Genes that typically participate in matrix remodeling and repair, such as arginase I, which governs proline/hydroxyproline production, and chitinase 3 like-2, are induced in this stage of microglial function. Inducible NOS (iNOS) activity is concomitantly lowered. We have shown that alternative activation genes are expressed in both mouse models of AD and in human brains with AD (Colton et al., 2006), confirming that microglia in chronic neurodegenerative diseases are likely to demonstrate significant functional heterogeneity that includes an alternative state.

The observed lack of amyloid plaque removal by microglia may be attributed to this altered functional state, where both protease production and phagocytic responses are likely to be different. It is clear that an additional stimulus is required to initiate microglial removal of amyloid. A series of studies on antibody therapy showed that anti-Aβ antibody administration, both intracranially and systemically, results in microglial activation and concomitant removal of compact amyloid plaques (Wilcock et al., 2003; 2004). Indeed, when microglial activation was inhibited by anti-inflammatory compounds or using F(ab’)2 fragment (thus avoiding Fc-receptor activation), no clearance of compact amyloid was seen (Wilcock et al., 2004). Application of intracranial injection of LPS, a well-known inducer of classical activation, was shown to reduce diffuse amyloid significantly but only temporarily, and this is consistent with the acute nature of LPS-mediated microglial stimulation (Dicarlo et al., 2001; Herber et al., 2004).

Together, these data suggest that microglia are ineffective in the removal of amyloid until a stimulus is available that alters their activation status from one of tissue remodeling to one of tissue defense.

References:
Vitek MP, Bhattacharya K, Glendening JM, Stopa E, Vlassara H, Bucala R, Manogue K, Cerami A. Advanced glycation end products contribute to amyloidosis in Alzheimer disease. Proc Natl Acad Sci U S A. 1994 May 24;91(11):4766-70. Abstract

Jarrett JT, Berger EP, Lansbury PT. The carboxy terminus of the beta amyloid protein is critical for the seeding of amyloid formation: implications for the pathogenesis of Alzheimer's disease. Biochemistry. 1993 May 11;32(18):4693-7. Abstract

Colton CA, Mott RT, Sharpe H, Xu Q, Van Nostrand WE, Vitek MP. Expression profiles for macrophage alternative activation genes in AD and in mouse models of AD. J Neuroinflammation. 2006 ;3():27. Abstract

Wilcock DM, DiCarlo G, Henderson D, Jackson J, Clarke K, Ugen KE, Gordon MN, Morgan D. Intracranially administered anti-Abeta antibodies reduce β-amyloid deposition by mechanisms both independent of and associated with microglial activation. J Neurosci. 2003 May 1;23(9):3745-51. Abstract

Wilcock DM, Rojiani A, Rosenthal A, Levkowitz G, Subbarao S, Alamed J, Wilson D, Wilson N, Freeman MJ, Gordon MN, Morgan D. Passive amyloid immunotherapy clears amyloid and transiently activates microglia in a transgenic mouse model of amyloid deposition. J Neurosci. 2004 Jul 7;24(27):6144-51. Abstract

Wilcock DM, Munireddy SK, Rosenthal A, Ugen KE, Gordon MN, Morgan D. Microglial activation facilitates Abeta plaque removal following intracranial anti-Abeta antibody administration. Neurobiol Dis. 2004 Feb ;15(1):11-20. Abstract

DiCarlo G, Wilcock D, Henderson D, Gordon M, Morgan D. Intrahippocampal LPS injections reduce Abeta load in APP+PS1 transgenic mice. Neurobiol Aging. 2001 Nov-Dec ;22(6):1007-12. Abstract

Herber DL, Roth LM, Wilson D, Wilson N, Mason JE, Morgan D, Gordon MN. Time-dependent reduction in Abeta levels after intracranial LPS administration in APP transgenic mice. Exp Neurol. 2004 Nov 1;190(1):245-53. Abstract

View all comments by Carol Colton
View all comments by Michael Vitek
View all comments by Donna Wilcock

Rapid Plaque Growth and Positive Cooperative Assembly
Amyloid-β peptides constitute an intriguing class of molecules that self-assemble into stable, ordered structures, and their formation is reminiscent of the natural phenomenon of positive cooperative assembly. In general, this cooperativity is regulated by an allosteric effect, so that interactive assemblies, once formed, support exponential rates of subsequent growth. In biology, this phenomenon is widely observed all the way from the atomic to the molecular level—from the cooperative binding of calcium ions regulating the intercellular adhesive actions of transmembrane cadherins (1) to the allosteric cooperativity of protein kinase A generated by nucleotide and substrate positioning (2).

Interestingly, allosteric cooperativity of ligand binding may be disrupted by single amino acid mutations, for example, the (Y204A) site change in protein kinase A, suggesting that relatively subtle changes in ligand topography abruptly attenuate the cooperativity mechanism. The addition to Meyer-Luehmann and...  Read more

Rapid Plaque Growth and Positive Cooperative Assembly
Amyloid-β peptides constitute an intriguing class of molecules that self-assemble into stable, ordered structures, and their formation is reminiscent of the natural phenomenon of positive cooperative assembly. In general, this cooperativity is regulated by an allosteric effect, so that interactive assemblies, once formed, support exponential rates of subsequent growth. In biology, this phenomenon is widely observed all the way from the atomic to the molecular level—from the cooperative binding of calcium ions regulating the intercellular adhesive actions of transmembrane cadherins (1) to the allosteric cooperativity of protein kinase A generated by nucleotide and substrate positioning (2).

Interestingly, allosteric cooperativity of ligand binding may be disrupted by single amino acid mutations, for example, the (Y204A) site change in protein kinase A, suggesting that relatively subtle changes in ligand topography abruptly attenuate the cooperativity mechanism. The addition to Meyer-Luehmann and colleagues’ innovative system of specific mutation-containing amyloid peptides or other interruptive molecules that do not support cooperative assembly may be an attractive pharmacological strategy to alter the kinetics of rapid plaque growth and the onset of Alzheimer neuropathology.

References:
1. Courjean O, Chevreux G, Perret E, Morel A, Sanglier S, Potier N, Engel J, Dorsselaer AV, Feracci H. Modulation of E-cadherin monomer folding by cooperative binding of calcium ions. Biochemistry. 2008 Jan 31; [Epub ahead of print] Abstract

2. Masterson LR, Mascioni A, Traaseth NJ, Taylor SS, Veglia G. Allosteric cooperativity in protein kinase A. Proc Natl Acad Sci U S A. 2008 Jan 15;105(2):506-11. Epub 2008 Jan 4. Abstract

View all comments by Walter J. Lukiw

This is a fascinating paper, which I will be presenting in a journal club soon. I am sure there will be many answers from future studies using this imaging technique. But already it is interesting to see the rapid formation of amyloid plaques in vivo.

I wonder, however, could plaque formation happen even more rapidly in brain? The fluorescence dye used in this paper is a derivative of congo red, which may interfere with amyloid fibril formation. In this case, it is possible that the kinetics in this paper is still an underestimate. One thing that puzzles me, as a former Alzheimer researcher, is that environmental enrichment is reported to increase the number of amyloid plaques in the hippocampus of APPswe/PS1d9 mice (Jankowsky et al., 2003 powID=33494), while the same treatment also improves their learning performance (Jankowsky et al., 2005 powID=45618). How does that fit into the model?

Nevertheless, this paper has created new ground, and I assume that the authors have even more longitudinal imaging data in hand by now, hopefully for several months.

View all comments by Hiroaki Misono

This landmark study provides many exciting new insights into the development of β amyloid plaques, and is a superb example of the importance of descriptive neuropathology research in elucidating Alzheimer disease (AD) pathogenesis. Using multiphoton microscopy to repeatedly image brain areas in transgenic mouse models of AD, the authors made several novel observations, including that plaques form within a day and remain stable in size, occur prior to microglial activation, and are not directly related to the vasculature. Another interesting new finding was that dystrophic neurites in plaque-free areas can appear and disappear.

The authors argue that their data indicate that plaques do not develop from dystrophic neurites, since plaques were not observed to form at sites of dystrophic neurites in plaque-free areas. Yet, looking closely at the brain cytoarchitecture prior to the appearance of a plaque, abundant neurites are evident, and with the limited resolution of multiphoton microscopy, early neuritic alterations could be missed spatially. They could also be missed...  Read more

This landmark study provides many exciting new insights into the development of β amyloid plaques, and is a superb example of the importance of descriptive neuropathology research in elucidating Alzheimer disease (AD) pathogenesis. Using multiphoton microscopy to repeatedly image brain areas in transgenic mouse models of AD, the authors made several novel observations, including that plaques form within a day and remain stable in size, occur prior to microglial activation, and are not directly related to the vasculature. Another interesting new finding was that dystrophic neurites in plaque-free areas can appear and disappear.

The authors argue that their data indicate that plaques do not develop from dystrophic neurites, since plaques were not observed to form at sites of dystrophic neurites in plaque-free areas. Yet, looking closely at the brain cytoarchitecture prior to the appearance of a plaque, abundant neurites are evident, and with the limited resolution of multiphoton microscopy, early neuritic alterations could be missed spatially. They could also be missed temporally, as supplementary Figure 4 shows that neuritic dystrophy can be transient. In addition, compared to the many imaging sessions required to find emergence of new plaques (only 26 new plaques were found imaging 1,285 times at 238 sites in 14 mice), it is not specified how often the authors looked for the formation of plaques at sites of dystrophic neurites; only 10 examples are mentioned. Given how infrequently plaque formation was captured overall, following only 10 examples does not seem definitive.

The authors show that neuritic dystrophy follows plaque formation. However, they also observe that dystrophic neurites occur in plaque-free areas, indicating that extracellular plaques are not required for dystrophic neurite formation. Kumar-Singh points out in his comment that a higher power imaging method to view early β amyloid accumulation and dystrophic neurites at an ultrastructural level would be interesting. In fact, such electron microscopy studies have been done, reporting early intracellular β amyloid accumulation and even oligomerization within dystrophic neurites and synaptic compartments in both areas with and without plaques in AD transgenic mouse and human AD brain.

The Alzforum news story also notes: "whether a newly formed plaque changes neural activity...remains to be tackled." In our EM studies, we have consistently noted that even in the absence of plaques, intracellular Aβ accumulates prominently in neurites and synaptic compartments. Moreover, intracellular accumulation of Aβ is associated with marked ultrastructural pathology. Based on this ultrastructural pathology, it seems highly unlikely that these neurites and synapses would be capable of normal synaptic function. Both axonal and dendritic transport is also unlikely to be normal.

Before the current study is taken as proof that only extracellular β amyloid plays a role in the formation of plaques and neuritic dystrophy, one might want to keep an open mind for a role also of intraneuronal Aβ. An alternative scenario is that accumulation of intraneuronal Aβ both causes neuritic dystrophy and can be the nidus for extracellular plaque formation.

View all comments by Estibaliz Capetillo-Zarate
View all comments by Gunnar Gouras
View all comments by Michael Lin

I wanted to thank Serge Rivest, Mathias Jucker, Tony Wyss-Coray, Joseph El Khoury, and Pritam Das for their helpful and thought-provoking comments, and to address some of their questions. I find it terribly interesting that the recent report by Richard, Rivest, and colleagues showed spontaneously increased TGF-β expression in immune cells near plaques of Tg APP/TLR2-/- mice. I agree that these striking findings are in line with the interpretation that increased TGF-β1 levels in AD patient brains, as shown by Wyss-Coray, Masliah, Mucke, and colleagues, likely serve the maladaptive role of maintaining an “immune privileged” brain milieu in AD patients and in these transgenic mouse models of the disease. We believe that overcoming this non-productive immune state will likely be key in targeting beneficial immune-mediated clearance of cerebral amyloid—and what better immune cell to target than the blood-borne macrophage (Greek etymology—“big eater”)? We also agree with Joseph El Khoury that a key aspect of this therapeutic modality will be promoting the Aβ...  Read more

I wanted to thank Serge Rivest, Mathias Jucker, Tony Wyss-Coray, Joseph El Khoury, and Pritam Das for their helpful and thought-provoking comments, and to address some of their questions. I find it terribly interesting that the recent report by Richard, Rivest, and colleagues showed spontaneously increased TGF-β expression in immune cells near plaques of Tg APP/TLR2-/- mice. I agree that these striking findings are in line with the interpretation that increased TGF-β1 levels in AD patient brains, as shown by Wyss-Coray, Masliah, Mucke, and colleagues, likely serve the maladaptive role of maintaining an “immune privileged” brain milieu in AD patients and in these transgenic mouse models of the disease. We believe that overcoming this non-productive immune state will likely be key in targeting beneficial immune-mediated clearance of cerebral amyloid—and what better immune cell to target than the blood-borne macrophage (Greek etymology—“big eater”)? We also agree with Joseph El Khoury that a key aspect of this therapeutic modality will be promoting the Aβ phagocytosis response while opposing the proinflammatory response, both of which likely exist as a continuum of innate immune cell activation profiles (Town et al., 2005). But, if we can accomplish this, will amyloid-reducing therapies ultimately be successful AD therapeutics? As stated by Dave Morgan and others on this forum, the first test of the amyloid cascade hypothesis of AD in humans will likely be the Aβ vaccine. We anxiously await whether the hypothesis holds up and delivers an efficacious AD therapy. If it does, then the floodgates will open for a whole host of amyloid-targeted AD therapeutics—both immune and non-immune.

About the issue raised by Mathias Jucker and Tony Wyss-Coray of CD11c as a marker for blood-borne innate immune cells/macrophages versus microglia, I should mention that we initially thought that CD11c would be a microglial marker in the context of AD. However, after examining numerous brain sections from various ages of wild-type versus Tg2576 or mutant APP/PS1 doubly transgenic mice for CD11c expression, we concluded that while microglia in the parenchyma around Aβ deposits were CD11b, CD45, MHC II, F4/80 Ag, and CD68 positive, they were negative for CD11c. However, we did observe a small number of round, non-process bearing CD11c positive cells within the lumen of blood vessels in both Tg2576 and APP/PS1 mice, consistent with Stalder and colleagues’ report of invading hematopoietic cells in brains of aged Tg2576 mice. At the time that we were checking for CD11c expression in AD mice, Alon Monsonego and Harold Weiner published a review in Science where they mentioned (as data not shown) that plaque-associated microglia were CD11c positive. I called Alon and asked him about the methodological details. However, after trying various tissue handling techniques, antibodies, and confocal settings, I was unable to reproduce this despite getting microglia in day 20 MOG-EAE brain sections to light up like a Christmas tree with CD11c. I came away thinking that it is possible to acutely activate microglia with the necessary vigor to promote CD11c expression, for example, in the context of EAE. However, I believe that this form of activation does not occur in AD mice, where the profile more closely resembles a chronic, persistent, low-level inflammation.

I have recently read the paper by Bulloch and coworkers with great interest, which shows the presence of CD11c/EYFP “dendritic-like” mouse microglia in multiple stages of life. However, because the authors did not quantify their observations, it is unclear how prevalent these cells are in the brain, and/or whether these cells arose from the blood or were long-term CNS residents. Further, the authors had difficulty in co-staining these cells with CD11c antisera in tissue sections, raising a possibility that those who work with transgenics are all too aware of: expression of transgenes is often more promiscuous than expected. In our study, we demonstrated a seven- to eightfold increase in CD45+CD11b+CD11c+CD68+Ly-6C- cells (presumed “anti-inflammatory” macrophages initially immunophenotyped by Littman’s group in Geissmann et al., 2003) in our crossed mice, and immunohistochemical approaches revealed prominent vascular cuffing, where these cells appeared to be entering the brain via cerebrovessels. Regarding the questions from Joseph El Khoury and Pritam Das about the origin of these brain macrophages, we agree that the “acid test” of whether the macrophage-like cells that we see in and around cerebral vessels and β amyloid plaques arise from the periphery or from within the CNS would either be a chimeric approach or parabiosis. We moved away from the chimeric approach following recent reports in Nature Neuroscience (Ajami et al., 2007; Mildner et al., 2007) showing that the act of irradiating the mice leads to brain infiltration of monocytes/macrophages—the very dependent variable that we are interested in testing. However, we believe that 1) parabiosis of AD mice with GFP+CD11c-DNR mice or 2) chemical methods of ablating hematopoietic cells in AD mice followed by reconstitution with GFP+CD11c-DNR bone marrow containing or depleted of macrophages represent possible strategies that we are currently pursuing.

Finally, Pritam Das raises the interesting questions of the long-term consequences of inhibiting TGF-β signaling on peripheral macrophages and the effects on T cells. We did not observe increased peripheral numbers of innate immune cells (including macrophages and dendritic cells), CD4+ or CD8+ T cells, or B cells in CD11c-DNR mice alone or in Tg2576xCD11c-DNR crossed mice, suggesting that an autoimmune state was not generated and that the increased abundance of macrophages in the brains of our crossed mice was β amyloid-directed. We also quantified T cells in brains of our crossed mice versus singly transgenic animals, and detected that about 4-5 percent of brain hematopoietic cells were TcRαβ positive (presumed T cells), and they were divided about equally between CD4+ and CD8+ subsets—however, these numbers were similar amongst wild-type, CD11c-DNR, APP/PS1, and APP/PS1xCD11c-DNR mice, suggesting that neither the CD11c-DNR nor the APP/PS1 transgenes were able to modify brain entry of T cells. Finally, regarding the issue of assessing neurodegeneration, we are currently pursuing this line of investigation by quantitative synaptophysin immunohistochemistry and hope to answer this question in the near future.

References:
Ajami B, Bennett JL, Krieger C, Tetzlaff W, Rossi FM. Local self-renewal can sustain CNS microglia maintenance and function throughout adult life. Nat Neurosci. 2007 Dec;10(12):1538-43. Abstract

Bulloch K, Miller MM, Gal-Toth J, Milner TA, Gottfried-Blackmore A, Waters EM, Kaunzner UW, Liu K, Lindquist R, Nussenzweig MC, Steinman RM, McEwen BS. CD11c/EYFP transgene illuminates a discrete network of dendritic cells within the embryonic, neonatal, adult, and injured mouse brain. J Comp Neurol. 2008 Jun 10;508(5):687-710. Abstract

Geissmann F, Jung S, Littman DR. Blood monocytes consist of two principal subsets with distinct migratory properties. Immunity. 2003 Jul;19(1):71-82. Abstract

Mildner A, Schmidt H, Nitsche M, Merkler D, Hanisch UK, Mack M, Heikenwalder M, Brück W, Priller J, Prinz M. Microglia in the adult brain arise from Ly-6C(hi)CCR2(+) monocytes only under defined host conditions. Nat Neurosci. 2007 Dec 1;10(12):1544-53. Abstract

Monsonego A, Weiner HL. Immunotherapeutic approaches to Alzheimer's disease. Science. 2003 Oct 31;302(5646):834-8. Abstract

Richard KL, Filali M, Préfontaine P, Rivest S. Toll-like receptor 2 acts as a natural innate immune receptor to clear amyloid beta 1-42 and delay the cognitive decline in a mouse model of Alzheimer's disease. J Neurosci. 2008 May 28;28(22):5784-93. Abstract

Stalder AK, Ermini F, Bondolfi L, Krenger W, Burbach GJ, Deller T, Coomaraswamy J, Staufenbiel M, Landmann R, Jucker M. Invasion of hematopoietic cells into the brain of amyloid precursor protein transgenic mice. J Neurosci. 2005 Nov 30;25(48):11125-32. Abstract

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

Wyss-Coray T, Masliah E, Mallory M, McConlogue L, Johnson-Wood K, Lin C, Mucke L. Amyloidogenic role of cytokine TGF-1 in transgenic mice and in Alzheimer's disease. Nature. 1997 Oct 9;389(6651):603-6. Abstract

View all comments by Terrence Town

I am glad that the researchers studying transgenic models are finally confirming our results published in 2002 (Fiala et al., 2002), which showed transmigration of macrophages across the brain vessel wall and clearance of plaques by these large macrophages.

The migrating macrophages broke through ZO-1 tight junction barrier and aggregated around brain vessels similarly as in HIV encephalitis. This has been followed by a recent publication in PNAS (Fiala et al., 2007). The animal studies cannot resolve the crucial question: are macrophages of patients with AD different from those of control subjects? The answers for interested readers are available in our PNAS article and more current work presented at ICAD. Not only macrophages penetrate across the blood-brain barrier but also clear oligomeric amyloid-β from neurons.

References:
Fiala M, Liu QN, Sayre J, Pop V, Brahmandam V, Graves MC, Vinters HV. Cyclooxygenase-2-positive macrophages infiltrate the Alzheimer's disease brain and damage the blood-brain barrier. Eur J Clin Invest. 2002 May;32(5):360-71. Abstract

Fiala M, Liu PT, Espinosa-Jeffrey A, Rosenthal MJ, Bernard G, Ringman JM, Sayre J, Zhang L, Zaghi J, Dejbakhsh S, Chiang B, Hui J, Mahanian M, Baghaee A, Hong P, Cashman J. Innate immunity and transcription of MGAT-III and Toll-like receptors in Alzheimer's disease patients are improved by bisdemethoxycurcumin. Proc Natl Acad Sci U S A. 2007 Jul 31;104(31):12849-54. Abstract

View all comments by Milan Fiala

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...  Read more

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

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...  Read more

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