Differential roles of cyclin-dependent kinase 5 in tangential and radial migration of cerebellar granule cells.

The cerebellar granule cell is a unique neuron which undergoes tangential migration along axonal tracts and radial migration along glial fibers sequentially during postnatal development. Little is known about molecular bases of the differential kinetics of tangential and radial migration. Here we developed a time-lapse imaging assay for tangential migration of cerebellar granule cells, and investigated comparative contributions of cyclin-dependent kinase 5 (CDK5), a key regulator of neuronal migration, in tangential and radial migration of granule cells in vivo and in organotypic cultures. Overexpression of a dominant-negative form of CDK5 severely disrupted cell morphology and somal movement during radial migration, while it only moderately affected tangential migration. Dominant-negative inhibition of CDK5 induced formation of ectopic radial processes in granule cells in vivo which aberrantly elongated into the white matter in the cerebellum. Live imaging of granule cell migration in cerebellar slices revealed that CDK5 regulates not only nuclear migration but also centrosome movement during radial migration. These findings suggest a mode-specific function of CDK5 in neuronal migration

ECHO-liveFISH: in vivo RNA labeling reveals dynamic regulation of nuclear RNA foci in living tissues.

生きたマウス脳内の細胞内RNA活動の可視化に成功 -早くて正確な製薬時のスクリーニングなど応用に期待-. 京都大学プレスリリース. 2015-06-29.Elucidating the dynamic organization of nuclear RNA foci is important for understanding and manipulating these functional sites of gene expression in both physiological and pathological states. However, such studies have been difficult to establish in vivo as a result of the absence of suitable RNA imaging methods. Here, we describe a high-resolution fluorescence RNA imaging method, ECHO-liveFISH, to label endogenous nuclear RNA in living mice and chicks. Upon in vivo electroporation, exciton-controlled sequence-specific oligonucleotide probes revealed focally concentrated endogenous 28S rRNA and U3 snoRNA at nucleoli and poly(A) RNA at nuclear speckles. Time-lapse imaging reveals steady-state stability of these RNA foci and dynamic dissipation of 28S rRNA concentrations upon polymerase I inhibition in native brain tissue. Confirming the validity of this technique in a physiological context, the in vivo RNA labeling did not interfere with the function of target RNA nor cause noticeable cytotoxicity or perturbation of cellular behavior

CTCF loss induces giant lamellar bodies in Purkinje cell dendrites

Abstract CCCTC-binding factor (CTCF) has a key role in higher-order chromatin architecture that is important for establishing and maintaining cell identity by controlling gene expression. In the mature cerebellum, CTCF is highly expressed in Purkinje cells (PCs) as compared with other cerebellar neurons. The cerebellum plays an important role in motor function by regulating PCs, which are the sole output neurons, and defects in PCs cause motor dysfunction. However, the role of CTCF in PCs has not yet been explored. Here we found that the absence of CTCF in mouse PCs led to progressive motor dysfunction and abnormal dendritic morphology in those cells, which included dendritic self-avoidance defects and a proximal shift in the climbing fibre innervation territory on PC dendrites. Furthermore, we found the peculiar lamellar structures known as “giant lamellar bodies” (GLBs), which have been reported in PCs of patients with Werdnig-Hoffman disease, 13q deletion syndrome, and Krabbe disease. GLBs are localized to PC dendrites and are assumed to be associated with neurodegeneration. They have been noted, however, only in case reports following autopsy, and reports of their existence have been very limited. Here we show that GLBs were reproducibly formed in PC dendrites of a mouse model in which CTCF was deleted. GLBs were not noted in PC dendrites at infancy but instead developed over time. In conjunction with GLB development in PC dendrites, the endoplasmic reticulum was almost absent around the nuclei, the mitochondria were markedly swollen and their cristae had decreased drastically, and almost all PCs eventually disappeared as severe motor deficits manifested. Our results revealed the important role of CTCF during normal development and in maintaining PCs and provide new insights into the molecular mechanism of GLB formation during neurodegenerative disease