Precise regulation of RNA metabolism is crucial for dynamic gene expression and controlling cellular functions. In the nervous system, defects in RNA metabolism are implicated in disturbing brain homeostasis and development. Here we report that deubiquitinating enzyme, ubiquitin specific peptidase 15 (USP15), deubiquitinates terminal uridylyl transferase 1 (TUT1) and changes global RNA metabolism. We found that the expression of USP15 redistributes TUT1 from nucleolus to nucleoplasm, resulting in the stabilization of U6-snRNA. We also found that lack of the Usp15 gene induces impairment in motor ability with the unconventional cerebellar formation. Moreover, inhibition of the USP15-TUT1 cascade triggered mild and chronic endoplasmic reticulum (ER) stress. Therefore, our results suggest that USP15 is crucial for mRNA metabolism and maintains a healthy brain. These findings provide a possibility that disturbance of the USP15-TUT1 cascade may induce chronic and mild ER stress, leading to an acceleration of neurodegenerative phenotype.

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In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, survival, and glial communications. To date, numerous studies have reported that defects in these systems cause various neuronal diseases. Thus, understanding the mechanisms underlying vesicle dynamics may provide influential clues that could aid in the treatment of several neuronal disorders. Here, we describe a method for quantifying vesicle motilities, such as motility distance and rate of movement, using a software plug-in for the ImageJ platform. To obtain images for quantification, we labeled neuronal endosome-lysosome structures with EGFP-tagged vesicle marker proteins and observed the movement of vesicles using a time-lapse microscopy. This method is highly useful and simplify measuring vesicle motility in neurites, such as axons and dendrites, as well as in the soma of both neurons and glial cells. Furthermore, this method can be applied to other cell lines, such as fibroblasts and endothelial cells. This approach could provide a valuable advancement of our understanding of membrane trafficking.

The micronucleus is known to be a biomarker for genomic instability, which is a hallmark of tumors and aging. Normally, micronuclei are produced by segregation errors and mechanical stresses arising from dividing or migrating cells, leading to activation of the innate immune response pathway. Although micronuclei often emerge in damaged tissues, the quantitative procedure for analyzing micronuclei accurately has been problematic. Here, we introduce a novel MATLAB-based program for quantifying micronuclei (CAMDi: calculating automatic micronuclei distinction) in vitro and in vivo. CAMDi is adaptable to various experimental imaging techniques and is useful for obtaining reproducible data. CAMDi enables us to measure the accurate size of micronuclei from the three-dimensional images. Using CAMDi, we revealed a novel link between the emergence of micronuclei and neuroinflammation. We found that inflammatory stimulation does not increase the number of micronuclei in primary neurons. On the other hand, the administration of lipopolysaccharide into mice slightly increases micronuclei formation in neurons of the hippocampus region. These findings demonstrate that neuronal micronuclei formations are induced by an inflammatory response in a non-cell-autonomous manner. We provide a novel tool, CAMDi, to quantify micronuclei and demonstrate that neuronal micronuclei are produced not only by the cell-autonomous process but also by the intercellular communication associated with neuroinflammation in vivo.

Microglia are resident macrophages that are critical for brain development and homeostasis. Microglial morphology is dynamically changed during postnatal stages, leading to regulating synaptogenesis and synapse pruning. Moreover, it has been well known that the shape of microglia is also altered in response to the detritus of the apoptotic cells and pathogens such as bacteria and viruses. Although the morphologic changes are crucial for acquiring microglial functions, the exact mechanism which controls their morphology is not fully understood. Here, we report that the FAT atypical cadherin family protein, FAT3, regulates the morphology of microglial cell line, BV2. We found that the shape of BV2 becomes elongated in a high-nutrient medium. Using microarray analysis, we identified that FAT3 expression is induced by culturing with a high-nutrient medium. In addition, we found that purinergic analog, hypoxanthine, promotes FAT3 expression in BV2 and mouse primary microglia. FAT3 expression induced by hypoxanthine extends the time of sustaining the elongated forms in BV2. These data suggest that the hypoxanthine-FAT3 axis is a novel pathway associated with microglial morphology. Our data provide a possibility that FAT3 may control microglial transitions involved in their morphologic changes during the postnatal stages in vivo .

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