KLHL7 promotes TUT1 ubiquitination associated with nucleolar integrity: Implications for retinitis pigmentosa.

Kelch-like protein 7 (KLHL7) is a component of Cul3-based Cullin-RING ubiquitin ligase. Recent studies have revealed that mutations in klhl7 gene cause several disorders, such as retinitis pigmentosa (RP). Although KLHL7 is considered to be crucial for regulating the protein homeostasis, little is known about its biological functions. In this study, we report that KLHL7 increases terminal uridylyl transferase 1 (TUT1) ubiquitination involved in nucleolar integrity. TUT1 is normally localized in nucleolus; however, expression of KLHL7 facilitates a vulnerability of nucleolar integrity, followed by a decrease of TUT1 localization in nucleolus. On the other hand, pathogenic KLHL7 mutants, which causes an onset of RP, have little effect on both nucleolar integrity and TUT1 localization. Finally, KLHL7 increases TUT1 ubiquitination levels. Taken together, these results imply that KLHL7 is a novel regulator of nucleolus associated with TUT1 ubiquitination. Our study may provide a valuable information to elucidate a pathogenic mechanism of RP.

CACUL1/CAC1 attenuates p53 activity through PML post-translational modification.

Promyelocytic leukaemia (PML) is a tumor suppressor protein covalently conjugated with SUMO family proteins, leading to the formation of PML nuclear bodies (NBs). PML-NBs provide a platform for efficient posttranslational modification of targets and protein-protein interaction, contributing to the adjustment of gene expression and chromatin integrity. Although PML SUMOylation is thought to play important roles in diverse cellular functions, the control mechanisms of adequate modification levels have remained unsolved. Here, we report that Cullin-related protein CACUL1/CAC1 (CACUL1) inhibits PML posttranslational modification. CACUL1 interacts with PML and suppresses PML SUMOylation, leading to the regulation of PML-NB size in the nucleus. We also found that Ubc9, a SUMO-conjugating enzyme, binds to CACUL1 and antagonizes the interaction between CACUL1 and PML. Furthermore, CACUL1 attenuates p53 transcriptional activity. These data suggest that CACUL1 is a novel regulator that negatively controls p53 activity through the regulation of PML SUMOylation.

Atypical Cadherin FAT3 Is a Novel Mediator for Morphological Changes of Microglia.

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.

USP15 Deubiquitinates TUT1 Associated with RNA Metabolism and Maintains Cerebellar Homeostasis.

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 the disturbance of 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 the nucleolus to nucleoplasm, resulting in the stabilization of U6 snRNA. We also found that lack of the Usp15 gene induces an impairment in motor ability with an 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 induces chronic and mild ER stress, leading to an acceleration of the neurodegenerative phenotype.

Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes.

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.