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

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