Incomplete acidification of lysosomes of microglia leads to limited degradation of internalized fAβ by these cells (Majumdar et al.
). In this article, we show that ClC-7 is inefficiently targeted to lysosomes in quiescent primary microglia. Instead, most ClC-7 in microglia is subject to degradation, apparently by an ERAD pathway. Transport of Cl−
ions can be important for the regulation of lysosomal pH (Ohkuma et al.
; Bae and Verkman, 1990
) because it helps to dissipate the inside-positive electrical potential that is generated by the electrogenic V-ATPase (Pillay et al.
; Graves et al.
). ClC-7 is the main Cl−
transporter in lysosomes (Graves et al.
), and evidence presented here indicates that its low levels in lysosomes of quiescent microglia is associated with impaired lysosomal acidification.
As noted in the Introduction
, our understanding of ion transport across the lysosomal membrane is incomplete, and other ion transport processes such as cation efflux have also been proposed to play an important role in regulating lysosomal pH (Steinberg et al.
). To examine the importance of counterion transport in facilitating acidification of microglial lysosomes, we used an experimental protocol in which the V-ATPase is inhibited by bafilomycin A1, and changes in lysosomal pH are then monitored when the proton-selective carrier CCCP is added to the cells (Lukacs et al.
). It is expected that the lysosomal pH should rise more quickly if there is sufficient counterion transport activity to balance the electrogenic efflux of protons mediated by CCCP. We found that addition of CCCP to quiescent microglia produced no measurable lysosome alkalinization in bafilomycin A1–treated quiescent microglia, but lysosome alkalinization was observed in the MCSF-activated microglia (Supplemental Figure S5). This result indicates that the lysosomes in MCSF-treated cells have greater counterion conduction than do lysosomes in quiescent microglia. The presence of ClC-7 could provide such an increase in counterion conductance, but other mechanisms could also contribute.
It is interesting to note that the lysosomal pH in microglia rose by ~0.7 pH units in 2 min when the cells were treated with bafilomycin A1. This rise was observed in both quiescent and MCSF-treated microglia (Supplemental Figure S5), and it indicates that there is a significant proton leak in the lysosomal membrane in microglia. By comparison, phagosomes in thioglycolate-elicited mouse macrophages alkalinized at a rate of 0.09 pH units per minute (Lukacs et al.
), and lysosomes in A431 cells alkalinized by 1 pH unit in 30–40 min (Yoshimori et al.
). The source of the proton leak will require further investigation. A further complicating factor in understanding lysosomal pH regulation in microglia is that signal transduction pathways can alter ion conductance; we found that acidification of lysosomes in MCSF-treated microglia is reduced by inhibition of protein kinase A (Majumdar et al.
Although the mechanisms of lysosomal pH regulation are not fully understood, it is clear that activated microglia have more acidic lysosomes than do quiescent microglia (Majumdar et al.
). Furthermore, this increased lysosomal acidification correlates strongly with the extent of ClC-7 recruitment to the lysosomes. Additionally, siRNA knockdown of ClC-7 in microglia reduces lysosomal acidification in MCSF-activated microglia. Taken together, these results establish ClC-7 as an important regulator of lysosomal acidification in microglia.
We investigated the mechanism behind the inefficient lysosomal delivery of ClC-7 in quiescent microglia. Ostm1 is the β subunit of ClC-7 (Lange et al.
), and it has been shown in other cells to be important for the ER exit and lysosomal trafficking of ClC-7 (Lange et al.
). We found that Ostm1 plays a critical role in lysosomal trafficking of ClC-7 in microglia. Quiescent microglia express a low level of Ostm1 protein, and the absence of adequate Ostm1 is associated with inefficient delivery of ClC-7 to lysosomes.
The degradation of ClC-7 is in many ways similar to ERAD-mediated turnover of other multisubunit membrane proteins, notably the α subunit of the heptameric T-cell receptor (TCR) complex (Lippincott-Schwartz et al.
; Vembar and Brodsky, 2008
). ERAD-mediated turnover of αTCR is mainly seen in immature T-cells, where synthesis of certain subunits of TCR is blocked. When T-cells mature, synthesis of all seven subunits of the TCR complex is initiated, which induces subunit interaction and rescues αTCR from ERAD-mediated degradation (Bonifacino et al.
In microglia, MCSF treatment increases the expression of both ClC-7 and Ostm1. Higher concentration of these two proteins enhances Ostm1/ClC-7 interaction and complex formation. Increased Ostm1/ClC-7 interaction would be expected to improve ER exit and lysosomal delivery of ClC-7. Interestingly, ClC-7 in primary microglia that is targeted for degradation becomes concentrated in the perinuclear region in a pattern similar to structures called aggresomes, which are cellular inclusions formed by misfolded proteins such as the cystic fibrosis mutant (Δ-508) CFTR (Kopito and Sitia, 2000
). The perinuclear accumulations of ClC-7 are not as compact as a mature aggresome formed by the cystic fibrosis mutant (Δ-508) CFTR (Kopito and Sitia, 2000
), but they appear similar to earlier stages of aggresome formation. Further work would be required to understand the detailed mechanisms for perinuclear segregation and degradation of the ClC-7 in quiescent microglia.
To demonstrate the relevance of our findings with respect to AD pathology, we observed ClC-7 localization in microglia surrounding Aβ plaques in tissue sections from a mouse model of AD. Most of the microglial cells around Aβ plaques have a diffuse staining pattern for ClC-7, which is unlike the punctate lysosomal staining pattern for ClC-7 seen in neurons. LAMP-1 staining for lysosomes showed a typical punctate pattern in the microglia that showed that ClC-7 in microglia in an AD mouse was not properly targeted to the lysosomes, similar to the observations in primary mouse microglia. Whereas ClC-7 in cultured primary microglia shows a perinuclear staining pattern, in adult microglia in brain tissue the ClC-7 distribution is diffuse and resembles an ER distribution. We do not understand the reason for this difference, but in both cases little ClC-7 gets to the lysosomes. Our finding that ClC-7 in microglia is not punctate is consistent with a previous observation of ClC-7 distribution in microglia in mouse brain slices (Wartosch et al.
Inefficient plaque degradation by microglia can explain the paradox of plaque persistence even with intense microglial recruitment (Bolmont et al.
). Studies in the (APPswe)/PS1 mouse models of AD have shown that treatment with MCSF leads to improvement in cognitive functions as analyzed by the water T-maze task (Boissonneault et al.
). Improvement of cognitive functions upon MCSF treatment was observed in mice that were at early and at late stages of the AD pathology (Boissonneault et al.
). Mice that showed improved cognitive functions after the MCSF treatment had a lower plaque burden and also demonstrated efficient plaque clearance by activated microglia (Boissonneault et al.
). Plaque clearance by activated microglia after MCSF treatment in the AD mouse model was due to the enhanced lysosomal degradation of amyloid β aggregates (Boissonneault et al.
), which correlates well with our studies with primary mouse microglia. This correlation suggests that the mechanism of pH regulation that we have described is likely to be relevant in vivo.
The data presented herein show that in microglia the delivery of ClC-7 to lysosomes affects lysosomal acidification. Studies with neurons, macrophages, and osteoclasts from the ClC-7−/−
mice, however, show that lysosomes can acidify to a pH near 5.0 by a mechanism that is independent of ClC-7 (Kornak et al.
; Kasper et al.
; Steinberg et al.
). The mechanisms regulating lysosomal acidification are complex and not fully understood. Different cell types might use different mechanisms to maintain a steady-state value of the lysosomal pH. One interesting point is that the steady-state value of the lysosomal pH in quiescent microglia is different from that of neurons, macrophages, or osteoclasts. Whereas these other cell types all have a lysosomal pH close to 5.0, quiescent microglia maintain their steady-state pH at 5.9. Furthermore, there is no evidence that these other cell types alter lysosomal pH in response to physiological stimuli. Thus it appears that the mechanisms of lysosomal pH regulation can vary among different cell types.
It is not clear what purpose is served by the activation-dependent acidification of microglial lysosomes. Dendritic cells also acidify their lysosomes upon activation as part of the process leading to antigen presentation (Trombetta et al.
). There is evidence that microglia, like dendritic cells, can engage in antigen presentation in the lymph nodes (Hochmeister et al.
), so control of pH in the lysosomes of microglia might also be associated with their role in antigen processing and presentation.
The failure of ClC-7 to traffic to the lysosomes is the main cause of the reduced lysosomal acidification in microglia. Increased lysosomal acidification and consequent fAβ degradation can be achieved if ClC-7 is properly assembled and targeted to the lysosomes. Our data provide a model () for the regulation of lysosomal pH in microglia during activation. We show that activation of microglia induces efficient ClC-7/Ostm1 complex formation, which leads to increased lysosomal acidification through the recruitment of ClC-7 to the lysosomes. These findings describe a novel mechanism for lysosomal pH regulation and also open up a paradigm for generating new therapeutic strategies for AD.
FIGURE 8: Model for lysosomal pH regulation in microglia. Model for the regulation of lysosomal pH in activated microglia. (A) In unactivated microglia ClC-7 is degraded by a pathway involving proteasomes that appears similar to ERAD quality control mechanisms (more ...)