The Secretion of miR-200s by a PKCζ/ADAR2 Signaling Axis Promotes Liver Metastasis in Colorectal Cancer

Most colorectal cancer (CRC)-related deaths are due to liver metastases. PKCζ is a tumor suppressor in CRC with reduced expression in metastasis. Given the importance of microRNAs (miRNAs) in regulating cellular plasticity, we performed an unbiased screening and identified the miR-200 family as the most relevant miRNAs downregulated by PKCζ deficiency. The regulation of the intracellular levels of miR-200 by PKCζ is post-transcriptional and involves their secretion in extracellular vesicles. Here, we identified ADAR2 as a direct substrate of PKCζ in CRC cells. Phosphorylation of ADAR2 regulates its editing activity, which is required to maintain miR-200 steady-state levels, suggesting that the PKCζ/ADAR2 axis regulates miR-200 secretion through RNA editing. Loss of this axis results in epithelial-to-mesenchymal transition (EMT) and increased liver metastases, which can be inhibited in vivo by blocking miR-200 release. Therefore, the PKCζ/ADAR2 axis is a critical regulator of CRC metastases through modulation of miR-200 levels.Research was supported by grants from the NIH ( R01DK108743 , R01CA172025 , and R01CA207177 to J.M.; R01CA192642 and R01CA218254 to M.T.D.-M.)

Changes of in vivo functional properties of microglia in aging and Alzheimer's disease

Dissertation ist gesperrt bis 22. Oktober 2022 !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

Healthy Brain Aging Modifies Microglial Calcium Signaling In Vivo

Brain aging is characterized by a chronic, low-grade inflammatory state, promoting deficits in cognition and the development of age-related neurodegenerative diseases. Malfunction of microglia, the brain-resident immune cells, was suggested to play a critical role in neuroinflammation, but the mechanisms underlying this malfunctional phenotype remain unclear. Specifically, the age-related changes in microglial Ca2+ signaling, known to be linked to its executive functions, are not well understood. Here, using in vivo two-photon imaging, we characterize intracellular Ca2+ signaling and process extension of cortical microglia in young adult (2⁻4-month-old), middle-aged (9⁻11-month-old), and old (18⁻21-month-old) mice. Our data revealed a complex and nonlinear dependency of the properties of intracellular Ca2+ signals on an animal’s age. While the fraction of cells displaying spontaneous Ca2+ transients progressively increased with age, the frequencies and durations of the spontaneous Ca2+ transients followed a bell-shaped relationship, with the most frequent and largest Ca2+ transients seen in middle-aged mice. Moreover, in old mice microglial processes extending toward an ATP source moved faster but in a more disorganized manner, compared to young adult mice. Altogether, these findings identify two distinct phenotypes of aging microglia: a reactive phenotype, abundantly present in middle-aged animals, and a dysfunctional/senescent phenotype ubiquitous in old mice