Abstract:
Microglial cells are the primary macrophages of the central nervous system and, as such, they provide the first line of immune defense in response to injury or disease. Malfunction of microglia was proposed to play a critical role in the development of age-related diseases such as Alzheimer’s disease (AD), and recently ‘omics’ studies have pointed to microglia as causal agents of the disease. The role of microglia in the context of AD and other age-related diseases, however, is far from being elucidated. First, we still lack information about the functional properties of these cells during normal aging. And second, we do not understand how the pathology (e.g. amyloid accumulation) interacts with the organism’s age. Detailed knowledge about cellular and physiological properties of microglia is therefore crucial, and this information can only be assessed by studying these cells in their native environment: the intact brain. In this work, we analyze for the first time the in vivo physiological changes of microglia during normal brain aging and provide new insights about their functional changes in AD by using high resolution two-photon imaging and two different approaches: i) characterization of the Ca2+ signaling properties of cortical microglia from mice during aging and AD and ii) characterization of microglia dynamics during homeostasis and AD. Our data revealed a bell-shaped relationship between the properties of the Ca2+ signals and the animal’s age, with the most frequent and largest Ca2+ transients observed in middle-aged (9-11 months old) mice, compared to young adult (2-4 months old) and old (18-21 months old) mice. Interestingly, we also found sex-specific changes in some of the Ca2+ signaling properties, such as in the fraction of spontaneously active microglia. Importantly, the reduction of the Ca2+ signaling activity in old microglia was accompanied by an impairment of their ATP-directed chemotactic properties. The changes observed in AD mice were different to those observed in normal aging, and were characterized by a higher fraction of spontaneously active microglia with significantly reduced amplitudes of the Ca2+ transients, compared to the age-matched WT mice. In this work, we also characterized for the first time the motility and turnover of microglia in young adult WT mice (i.e. during homeostasis) and AD pathology, by establishing a new labeling method for red (R), green (G) and blue (B) color coding of microglia based on miRNA9-regulated lentiviral vectors. By combining this approach with in vivo two-photon imaging, we characterized the migration, proliferation and death of cortical microglia. Our results revealed that, in the healthy young adult brain, microglia display a daily average migration rate of 1%, with a median translocation distance of ~20 µm. The migration pattern is saltatory, characterized by fast translocation alternating with long stationary periods. In addition, our results from WT mice revealed a high fraction of blood-vessel associated migration (along or toward blood vessels) and low turnover rates. Although in young adult AD mice the daily average migration rate was slightly higher compared to age-matched WT mice, we neither observed significant differences in their pattern, nor in the median speed or cumulative translocation distance. The proliferation and death rates were, however, significantly higher than those from WT mice, leading to significantly lower survival rates of microglia from AD mice. Accordingly, amyloid plaques harbored most of the dynamic microglia but also triggered the highest death rates. Taken together, this work provides i) a better understanding of the physiological changes in microglia during normal aging and AD, and ii) a versatile toolkit for studying individual live microglia, laying the ground for future analysis in other pathological conditions.
Microglia