The Brain and Microglial Recruitment—Think Local, Not Global

Microglia play an important, if controversial, role in Alzheimer disease (AD). While these phagocytic cells clear up deposits of amyloid-β (Aβ), they may also unleash a horde of cytokines harmful to neurons. On top of that, there is evidence that not all microglia are created equal. Those derived from the blood stream seem to be particularly adept at clearing Aβ from the brain (see ARF related news story), but there has been a nagging question about how much peripherally recruited cells contribute to the CNS pool of microglia. Not much, according to two papers published in the November 18 Nature Neuroscience online.

The problem with studies suggesting that brain microglia are derived from peripheral cells is that they commonly depend on detection of transplanted bone marrow-derived cells in the brain of irradiated recipient mice. That irradiation can lead to disruption of the blood-brain barrier, allowing cells normally restricted to the periphery to enter the brain, suggest researchers lead by Fabio Rossi at the University of British Columbia, Vancouver. That point is echoed by researchers in Germany led by Marco Prinz at the University of Gottingen. Both groups now show that in the absence of irradiation, none of the microglia found in the brain are recruited from the blood.

Rossi and colleagues circumvented the need for irradiation by using parabiosis, a technique where the circulatory systems of two genetically identical mice are shared. With one mouse expressing hematopoietic cells labeled with green fluorescent protein, the researchers were able to look for infiltrating GFP-positive microglia in the brain of the parasymbiotic partner under a variety of conditions.

First author Bahareh Ajami and colleagues found that in over 30 different sections taken from the brain, brainstem, and spinal cord of each mouse, not one GFP+ microglia was detected despite the presence of more than 2,000 cells that stained for the microglial marker Iba-1. The authors also failed to detect infiltration of peripheral microglia into the CNS in models of acute and chronic neurodegeneration and microgliosis. In one model, they severed the facial motor nerve after it emerges from the CNS. This typically results in acute local microgliosis, but when Ajami and colleagues carried out this procedure in one of the parasymbiotic pairs, they again failed to find infiltration of GFP+ microglia from the partner, suggesting that the gliosis is due to recruitment of local, CNS-based cells. For their chronic model, they coupled a normal mouse to an ALS mouse expressing mutant superoxide dismutase (mSOD). Again, despite rampant microgliosis in the spinal cord of the ALS parasymbiont, none of the cells detected were GFP-positive. As controls, the researchers used irradiated animals transplanted with hematopoietic bone marrow cells. In both the acute and chronic model, they found that the microglia surrounding the damaged neurons were derived from the transplant.

“Taken together, these result indicated that the replacement of microglia by circulating precursors can be induced by the experimental manipulations associated with irradiation and transplantation, but this replacement does not take place under physiological conditions,” write the authors. They tested this idea by irradiating the GFP+ symbiont partner and then carrying out the facial nerve axotomy 5 weeks later. In this case they found that 80 percent of the infiltrating microglia were GFP-positive, supporting the idea that irradiation, or perhaps some other type of damage to the BBB, is a prerequisite for peripheral microglial infiltration into the CNS.

Prinz and colleagues arrive at a similar conclusion but from a different direction. Because circulating monocytes have recently been shown to be heterogenous, first author Alexander Mildner and colleagues were interested in exactly which blood monocytes are the precursors of microglia. Monocytes fall into two types based on high or low expression of the cell-surface maker Ly-6C. The researchers found that both high expression of Ly-6C and expression of the chemokine receptor CCR2 are essential for infiltration of microglia into the brain following irradiation and bone marrow transplant (CCR2 was recently shown to be crucial for microglial clearance of Aβ in APP transgenic mice—see ARF related news story). But does this infiltration depend on a damaged blood-brain barrier?

To answer that, Mildner and colleagues used a highly focused beam to protect the brain from irradiation, then, 2 weeks following bone marrow transplantation, they measured microglial infiltration. They found that the protected brains were devoid of Ly-6Chi/CCR2+/+ donor-derived microglia. To check if infiltration might require a trigger, such as CNS damage, the authors carried out a similar experiment but this time using the neurotoxic agent cuprizone to induce demyelination in the corpus callosum. Again, in the mice whose brains were protected from radiation, the scientists found no infiltration. In animals that had full body radiation, the CCR2+/+ microglia did infiltrate the corpus callosum, indicating that Ly-6Chi/CCR2+ monocytes were recruited to the site of damage. “The data indicate that Ly-6Chi monocytes are only recruited to the demyelinating lesion when the brain was irradiated,” write the authors. Similarly, using the same facial nerve axotomy protocol employed by Rossi’s group, Mildner and colleagues found no infiltration of microglia to the site of damage if the animals’ brains were initially protected from radiation damage.

Do these findings spell the death of the infiltrating microglia hypothesis? Not if you ask Serge Rivest of Laval University, Quebec (see Simard et al., 2006). Rivest sounded a cautionary note about parabiosis in an e-mail to Alzforum. “The technique employed may not allow adequate chimerization, and GFP cells are just not able to compete with resident myeloid cells," he suggested in reference to the work from Rossi’s group. And even if microglial infiltration does depend on damage to the blood-brain barrier, peripheral microglia or their precursor could still be physiologically relevant to the brain. There is some evidence that the BBB is compromised in AD (see ARF related news story): stroke is a typical example of how circulating monocytes can gain entry into the CNS.—Tom Fagan

Comments

  1. The protean and itinerant nature of phagocytes has compelled researchers to devise increasingly ingenious experiments to establish their role in brain disorders, as exemplified nicely by the studies of Mildner et al. and Ajami et al. These researchers make a reasonably compelling case that significant infiltration of the brain by peripheral, bone marrow-derived macrophages requires a weakening of normal host barriers. Since phagocytes exist on both sides of the cerebrovascular wall, does it matter to the brain where the cells come from? I think it does; there is growing evidence for functional specialization in otherwise similar-appearing macrophages, and (from an evolutionary perspective) why would the brain be endowed with such an effective—and selective—obstacle to circulating phagocytes if their pedigree was unimportant?

    Regarding the enduring discussion of the role of phagocytes in Alzheimer disease, one additional issue—cerebral β amyloid angiopathy (CAA)—is worth a comment. The degree of CAA in Alzheimer disease is highly variable, but affected vessels can be appreciably impaired, as evidenced by an elevated risk of hemorrhagic stroke. It is therefore conceivable that CAA might augment the infiltration of circulating monocytes into the brain, thereby modifying the pathologic signature and course of disease. On the flip side, the presence of CAA could reflect subtle functional differences in brain phagocytes. El Khoury et al., 2007 found that the disruption of microglial accumulation via Ccr2 deficiency causes the early appearance of CAA and microhemorrhage in APP-transgenic mice. Phagocytes thus may help to regulate the compartmentalization of Aβ aggregates in brain, suggesting that the presence and phenotype of these cells can influence the risk of CAA in older humans.

    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. Nat Med. 2007 Apr;13(4):432-8. PubMed.

    View all comments by Lary Walker

  2. This is a very interesting paper. It presents compelling evidence that when the blood-brain barrier is damaged, Ly-6hiCcr2+ monocytes are direct circulating precursors of microglia in the blood. This is the most convincing evidence so far that a specific subset of circulating monocytes can develop into microglia.

    The implications for Alzheimer disease remain to be seen. Our own recent data clearly shows that in APP transgenic mice, early microglial accumulation in the brain is Ccr2 dependent, and several of these cells have surface characteristics of blood monocytes. The blood-brain barrier in AD mouse models (and likely in AD) is far from intact functionally (Dickstein et al., 2006) as it allows the influx of antibodies (Bard et al., 2000) and of circulating Aβ from the blood into the brain (Deane et al., 2003). In addition, in-vitro data using a model for the BBB indicate that interaction of Aβ42 with monocytes or endothelial cells on the brain side potentiated monocyte transmigration from the blood side to the brain side (Fiala et al., 1998; Giri et al., 2000). A possible scenario is that in AD, the “damaged” or activated BBB cells (perhaps as a result of Aβ deposition) facilitate the passage of Ly-6hiCCR2+ monocytes from the blood into the brain and the subsequent accumulation of these cells, which will ultimately differentiate into microglia. In support of this scenario, we found that in the absence of Ccr2, the initial site for Aβ accumulation is around blood vessels (El Khoury et al., 2007).

    I believe these findings illustrate that much work is further needed to fully understand the role of microglia in AD and how they accumulate in the brain in response to amyloid deposition.

    References:

    Dickstein DL, Biron KE, Ujiie M, Pfeifer CG, Jeffries AR, Jefferies WA. Abeta peptide immunization restores blood-brain barrier integrity in Alzheimer disease. FASEB J. 2006 Mar;20(3):426-33. PubMed.

    Bard F, Cannon C, Barbour R, Burke RL, Games D, Grajeda H, Guido T, Hu K, Huang J, Johnson-Wood K, Khan K, Kholodenko D, Lee M, Lieberburg I, Motter R, Nguyen M, Soriano F, Vasquez N, Weiss K, Welch B, Seubert P, Schenk D, Yednock T. Peripherally administered antibodies against amyloid beta-peptide enter the central nervous system and reduce pathology in a mouse model of Alzheimer disease. Nat Med. 2000 Aug;6(8):916-9. PubMed.

    Deane R, Du Yan S, Submamaryan RK, LaRue B, Jovanovic S, Hogg E, Welch D, Manness L, Lin C, Yu J, Zhu H, Ghiso J, Frangione B, Stern A, Schmidt AM, Armstrong DL, Arnold B, Liliensiek B, Nawroth P, Hofman F, Kindy M, Stern D, Zlokovic B. RAGE mediates amyloid-beta peptide transport across the blood-brain barrier and accumulation in brain. Nat Med. 2003 Jul;9(7):907-13. PubMed.

    Fiala M, Zhang L, Gan X, Sherry B, Taub D, Graves MC, Hama S, Way D, Weinand M, Witte M, Lorton D, Kuo YM, Roher AE. Amyloid-beta induces chemokine secretion and monocyte migration across a human blood--brain barrier model. Mol Med. 1998 Jul;4(7):480-9. PubMed.

    Giri R, Shen Y, Stins M, Du Yan S, Schmidt AM, Stern D, Kim KS, Zlokovic B, Kalra VK. beta-amyloid-induced migration of monocytes across human brain endothelial cells involves RAGE and PECAM-1. Am J Physiol Cell Physiol. 2000 Dec;279(6):C1772-81. PubMed.

    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.

    View all comments by Joseph El Khoury

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References

News Citations

  1. Calling for Backup: Microglia from Bone Marrow Fight Plaques in AD Mice
  2. Microglia—Medics or Meddlers in Dementia
  3. Merck Symposium: Surmounting the Blood-brain Barrier in Dementia Research

Paper Citations

  1. 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. PubMed.

Further Reading

Primary Papers

  1. 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. PubMed.
  2. 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-6ChiCCR2+ monocytes only under defined host conditions. Nat Neurosci. 2007 Dec;10(12):1544-53. PubMed.

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