INTRAVITAL PH IMAGING WITH THE NEW SENSOR APHID REVEALS INEFFICIENT LYSOSOMAL ACIDIFICATION IN MICROGLIA VERSUS INFLAMMATION-INDUCED ACIDIFICATION IN BRAIN MACROPHAGES

The linkage of lysosomal deficits to inflammation, aging and neurodegenerative diseases has been firmly established, but bridging published evidence in cell culture and biological tissues to animal models in vivo has been more challenging. To address that, we developed a methodology to image macrophage and microglia lysosomes and quantify their pH in vivo by intravital imaging using our new pH-sensitive probe ApHID (Sole-Domenech et al., 2025). With this new tool, we measured an average lysosomal pH in brain microglia of ~5.6 (with some compartments exhibiting a pH >6). This is consistent with the inefficient lysosomal acidification seen in cultured primary microglial cells, as in the absence of pro-inflammatory stimuli, primary microglial cells do not acidify their lysosomes efficiently. Moreover, using ApHID intravitally we also observed a progressive acidification of dural brain macrophages and infiltrating phagocytic cells present in brain areas subjected to pro-inflammatory ischemic lesion, demonstrating for the first time a direct link between inflammation and lysosomal acidification in vivo.
Because Alzheimer’s amyloid-beta (Aβ) deposits –a source of neurotoxic components– are resistant to degradation, and require full lysosomal enzymatic activity (at pH ~4.7) for complete proteolysis, we are now investigating lysosomal acidification in aging and Alzheimer’s Aβ brain pathology mouse models. Overall, our intravital methodology provides a new lens through which to study brain lysosomal biology during neuroinflammation and in brain disorders in which lysosome dysfunction plays a role.