Immunotherapy against Aβ leads to a striking removal of amyloid plaques in APP transgenic mice (Schenk et al., 1999
), and there is good evidence that microglia play an important role in this clearance (Bard et al., 2000
). However, the mechanisms by which immunization promotes clearance and degradation by microglia are uncertain. Recent studies have shown that activation of microglia in the absence of antibodies can also lead to the clearance of Aβ plaques in a mouse model of AD (Frenkel et al., 2005
). This indicates that antibodies to Aβ are not necessary for clearance of plaques, but it has been unclear how microglial activation or inflammatory stimuli can lead to improved clearance.
The data presented herein show that increased acidification of lysosomes may be the key factor that determines the ability of microglia to degrade fAβ. Microglia constitutively express a scavenger receptor, SRA, that can mediate the binding and uptake of fAβ (El Khoury et al., 1996
; Paresce et al., 1996
). As shown here, microglia have sufficient levels of lysosomal proteases to degrade proteins effectively; moreover, the levels of most proteases are higher in microglia than in J774 cells, which can digest fAβ efficiently. However, microglia have weakly acidic lysosomes, which would decrease the activity of many lysosomal proteases and could lead to the inability to degrade fAβ effectively. Treatment with inflammatory agents acidifies microglial lysosomes and makes them able to degrade fAβ. Although treatment with inflammatory agents can cause many changes in microglia, it is striking that a pulse-wash treatment with a weak base that transiently acidifies lysosomes also facilitates significant degradation of fAβ. This suggests that lysosomal acidification is the key event that is required for fAβ degradation by microglia. This phenomenon of increased proteolytic power as a consequence of lysosomal acidification was also described in dendritic cells during their maturation (Trombetta et al., 2003
In the assays of fAβ degradation with the ammonia pulse-wash or the forskolin treatment, we could only transiently acidify the lysosomes. In our experiments, the lysosomes were acidified four times for about an hour each time during a 72-h degradation assay. Nevertheless, we were able to observe ~40% degradation of fAβ as a consequence of these treatments. We typically observe <40% degradation in a 4-h chase even with macrophage cells (data not shown). It is possible that some key proteolytic enzymes are converted from the proenzyme to the active enzyme during the periods of high acidity and that these enzymes remain active during periods of reduced acidity in the microglial lysosomes.
We explored the mechanisms of lysosomal acidification in microglia. Several signal transduction pathways are activated in cells in response to MCSF treatment (Pixley and Stanley, 2004
). Activation of PKA by forskolin caused a drop in lysosomal pH similar to that seen in MCSF-treated cells. The acidification caused by forskolin was inhibited by treatment with DIDS, suggesting that anion transport was required for the acidification. These results would be consistent with activation of chloride channels downstream of PKA activation as a mechanism to lower the lysosomal pH in microglia. The lysosomal acidification in MCSF-treated microglia was partially reversed by inhibition of PKA or by treatment with DIDS, again indicating that PKA-dependent activation of chloride channels is important for the regulation of lysosomal acidification of microglia during inflammatory activation. Further work will be required to analyze the signaling pathways involved and to identify the regulated chloride channels on lysosomes of microglia.
There is evidence that immunization of AD model mice against Aβ leads to some reversal of behavioral impairments in addition to clearing amyloid deposits (Morgan et al., 2000
; Hartman et al., 2005
), and this suggests that this type of therapy might be beneficial. A major limitation is that inflammatory responses to immunization may be difficult to control, and a human trial of Aβ immunization had to be discontinued due to development of meningoencephalitis in some patients (Nicoll et al., 2003
It is possible that treatments that cause a specific type of microglial activation, leading to increased lysosomal acidification, may be especially beneficial in promoting clearance of plaques. It should be noted that not all types of microglial activation are equally effective. We found that treatment with MCSF, which causes microglia to express macrophage-like surface proteins, was especially effective at lowering lysosomal pH and activating the degradation of fAβ. In contrast, it has been reported that interaction with CD40 ligand can induce microglia to become more like antigen-presenting cells with decreased ability to phagocytose Aβ (Townsend et al., 2005
). Another report showed that activation of microglia via the IL-1 pathway might not be important for plaque clearance because IL-1 receptor knockout mouse cleared fAβ deposits after receiving immunotherapy (Das et al., 2006
). Other factors may also modulate the ability of microglia to internalize and degrade amyloid plaques. A recent study found that only bone marrow derived microglia and not resident microglia were capable of clearing fAβ deposits from the brains of AD model mice (Simard et al., 2006
In summary, we show that unactivated microglia do not degrade fAβ because they have weakly acidic lysosomes, and lysosomal acidification is a key downstream event leading to fAβ degradation by activated microglia. This may also be a key to the effectiveness of therapies to promote clearance of amyloid plaques by microglia, and treatments that acidify microglial lysosomes while minimizing other inflammatory reactions may be particularly beneficial.