Circulating monocytes, macrophages, and brain-resident microglia have all been implicated as responders to Alzheimer disease pathology, but exactly how these cells affect disease progression and whether circulating cells can even gain entry into the brain has been controversial. At this year’s Keystone Symposium called Alzheimer’s Disease Beyond Aβ, held January 10-15 at Copper Mountain, Colorado, researchers tried to make sense of the different cells and chemokine signaling pathways that might make the difference between quietly clearing Aβ and setting off a pro-inflammatory cascade that exacerbates pathology (see also related Copper Mountain news). One of the fractious signaling pathways seems to begin with activation of the fractalkine receptor (CX3CR1).
Fractalkine, the ligand, is located on the surface of endothelial cells in the periphery and on neurons in the brain. It is released from these cells after cleavage by ADAM10, or α-secretase (see Hundhausen et al., 2003). CX3CR1 is a chemokine receptor that, in the central nervous system, seems to be exclusively located on microglia (see Harrison et al., 1998). Work from Richard Ransohoff’s lab at the Cleveland Clinic, Ohio, also suggests that this receptor always co-localized with glial markers, such as Iba. At Copper Mountain, Ransohoff reported on some of his studies with fractalkine receptor knockout mice. These animals, engineered at Howard Hughes Investigator Dan Littman’s lab at New York University, appeared to have normal monocyte responses to peritonitis, peripheral antigen challenge, and peripheral nerve injury (see Jung et al., 2000), but Ransohoff wondered if there might be consequences in the brain of deleting CX3CR1.
Ransohoff reported that CX3CR1-/- mice are more susceptible to neurotoxicity after various central nervous system insults (see Cardona et al., 2006). They develop greater neuronal damage when challenged with lipopolysaccharide, which causes an inflammatory immune response. They are more susceptible to Parkinsonism, losing three times more dopaminergic neurons in the substantia nigra brain region when exposed to the mitochondrial toxin MPTP. The loss of fractalkine signaling also exacerbated toxicity in a mouse model of amyotrophic lateral sclerosis. Transgenic mice expressing the G93A human superoxide dismutase 1 lost more spinal cord neurons as one or both fractalkine receptor genes were knocked out. Their muscles also weakened faster. In these scenarios, Ransohoff and colleagues saw minimal recruitment of monocytes from the periphery, suggesting that brain-resident microglia, the only CNS cells that express CX3CR1, are responsible for the effects. Adoptive transfer studies supported this hypothesis. The researchers stereotactically injected the brain of wild-type mice with activated microglia prepared from the brains of heterozygous or homozygous CX3CR1 knockout mice. Thirty six hours after this adoptive transfer, only mice that received the CX3CR1 knockout microglia exhibited signs of neurotoxicity. Overall, the findings suggest that CX3CR1 expression prevents brain microglia from mounting a toxic response.
But does activation of CX3CR1 play a role in Alzheimer disease? This is a separate question, and to address it, Ransohoff, together with Cleveland Clinic colleagues Bruce Lamb, Kiran Bhaskar and students Sungho Lee and Nick Varvel, crossed the fractalkine receptor knockout mice with transgenic animals expressing human APP and presenilin1 and also with mice expressing human tau. In the latter case, the loss of the CX3CR1 receptor seemed detrimental. The researchers found greater tau hyperphosphorylation and more neurofibrillary tangles (as judged by immunohistochemistry) in htau/CX3CR1 KO compared to htau controls. Silver staining also detected more tau aggregates in the receptor knockouts. Why tau is more hyperphosphorylated when CX3CR1 is missing is not yet clear, but Ransohoff reported that p38MAP kinase activity increases in the fractalkine receptor’s absence and that blocking the kinase prevented tau hyperphosphorylation in the crosses. To probe what might energize the kinase, he used a trans-well co-culture system to grow primary cortical neurons and glia in the same medium without having them physically touch each other. With that, he found that CX3CR1-/- glia induce p38MAPK activity in the neurons, suggesting that some soluble factor mediates the response. Ransohoff said his lab is currently trying to figure out what that factor might be. The answer could be important since this particular kinase is linked to AD pathology in multiple ways. It is more active in AD brains compared to controls (see Sheng et al., 2001). It mediates Aβ’s suppression of long-term potentiation, which is crucial for proper learning and memory (see ARF related news story). And the kinase also has a propensity to set off pro-inflammatory cytokines (see Munoz et al., 2007 [add citation]). All of this had led to active pursuit of p38MAPK inhibitors for the potential treatment of AD (for a review, see Munoz and Ammit, 2010).
Ransohoff’s data suggest that microglia with a competent fractalkine response can prevent tau turning toxic. So Ransohoff said he was surprised to find that the receptor seems to have the opposite effect on Aβ. APP/PS1 mice with only one copy of CX3CR1 had fewer plaques compared to APP/PS1 control mice, while CX3CR1-negative transgenic mice had even less. APP processing seems identical in the three different mice, suggesting that the difference between them may lie in Aβ clearance, said Ransohoff. Interestingly, he reported finding fewer microglia surrounding plaques in wild-type mice compared to the chemokine receptor knockouts, which may indicate that CX3CR1 mutant microglia more actively clear plaques, he suggested.
What do the apparently opposing effects of CX3CR1 signaling on Aβ and tau pathology mean for Alzheimer disease? Some clues came from a talk by Joseph El Khoury, Massachusetts General Hospital, Charlestown. El Khoury has tried the same approach crossing APP/PS1 mice with CX3CR1 knockouts, and in support of Ransohoff’s findings, he reported that mice lacking the receptor had less soluble Aβ as detected by ELISA, and reduced Aβ deposition as judged by immunohistochemistry. The APP/PS1/CX3CR1-negative animals also performed significantly better than controls in the Barnes maze test of spatial learning and memory. Interestingly, fewer microglia surround plaques in these crosses.
All told, the data suggest that the role played by the fractalkine receptor in AD may be more complex than in PD and ALS, where it seems wholly protective. While it may protect against tau toxicity htau mice, the chemokine pathway seems to exacerbate plaque pathology (though Ransohoff cautions that both tau and Aβ effects have not been seen in the same mouse model). Why knocking out the receptor and reducing microglia activation, as reported by El Khoury, protects against plaques and improves memory is not clear, but El Khoury thinks it may be related to monocytes that phagocytose Aβ in vivo. He showed a video taken via a two-photon microscope trained on a transcranial window. In the video, GFP-labeled monocytes could be seen hovering around and plucking up Aβ deposits. Whether those monocytes could be persuaded to do the same thing in Alzheimer disease, for example by blocking fractalkine signaling, might be worth exploring.—Tom Fagan.
Part 1 of a two-part series. See Part 2.
- Copper Mountain: Origins and Actions of Glial Cells in AD
- Aβ Oligomers and Synaptic Dysfunction—Blame It on RAGE?
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- Sheng JG, Jones RA, Zhou XQ, McGinness JM, Van Eldik LJ, Mrak RE, Griffin WS. Interleukin-1 promotion of MAPK-p38 overexpression in experimental animals and in Alzheimer's disease: potential significance for tau protein phosphorylation. Neurochem Int. 2001 Nov-Dec;39(5-6):341-8. PubMed.
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