Wake-sleep cycles are severely disrupted by diseases affecting cytoplasmic homeostasis.

The circadian clock is based on a transcriptional feedback loop with an essential time delay before feedback inhibition. Previous work has shown that PERIOD (PER) proteins generate circadian time cues through rhythmic nuclear accumulation of the inhibitor complex and subsequent interaction with the activator complex in the feedback loop. Although this temporal manifestation of the feedback inhibition is the direct consequence of PER's cytoplasmic trafficking before nuclear entry, how this spatial regulation of the pacemaker affects circadian timing has been largely unexplored. Here we show that circadian rhythms, including wake-sleep cycles, are lengthened and severely unstable if the cytoplasmic trafficking of PER is disrupted by any disease condition that leads to increased congestion in the cytoplasm. Furthermore, we found that the time delay and robustness in the circadian clock are seamlessly generated by delayed and collective phosphorylation of PER molecules, followed by synchronous nuclear entry. These results provide clear mechanistic insight into why circadian and sleep disorders arise in such clinical conditions as metabolic and neurodegenerative diseases and aging, in which the cytoplasm is congested.

Streamlined procedure for gene knockouts using all-in-one adenoviral CRISPR-Cas9.

CRISPR-Cas9 is a powerful gene editing technique that can induce mutations in a target gene of interest in almost any mammalian cell line. However, its practicality can be limited if target cell lines are difficult to transfect and do not proliferate. In the current study, we have developed a streamlined approach for CRISPR-based gene knockouts with three key advantages, which allows phenotypic assay of gene knockouts without clonal selection and expansion. First, it integrates into a single, all-in-one vector transgenes for Cas9, sgRNA, and a fluorescence marker. Second, we used the Gateway system to rapidly clone specific sgRNAs into the all-in-one vector through PCR and in vitro recombination, without conventional enzyme digestion and ligation. Third, it uses adenovirus for the capacity to package the all-in-one vector, and for its high efficiency of transduction. We tested the all-in-one adenoviral CRISPR-Cas9 in a circadian clock model cell line U2OS, and demonstrated that essential clock genes such as Bmal1 and Per1 were knocked out so efficiently that functional assays could be performed from the heterogenic population without any clonal selection and expansion. This streamlined approach may prove invaluable for rapid functional assays of candidate genes in diverse biological pathways, including the circadian clock.

Microvascular endothelial cells engulf myelin debris and promote macrophage recruitment and fibrosis after neural injury.

The clearance of damaged myelin sheaths is critical to ensure functional recovery from neural injury. Here we show a previously unidentified role for microvessels and their lining endothelial cells in engulfing myelin debris in spinal cord injury (SCI) and experimental autoimmune encephalomyelitis (EAE). We demonstrate that IgG opsonization of myelin debris is required for its effective engulfment by endothelial cells and that the autophagy-lysosome pathway is crucial for degradation of engulfed myelin debris. We further show that endothelial cells exert critical functions beyond myelin clearance to promote progression of demyelination disorders by regulating macrophage infiltration, pathologic angiogenesis and fibrosis in both SCI and EAE. Unexpectedly, myelin debris engulfment induces endothelial-to-mesenchymal transition, a process that confers upon endothelial cells the ability to stimulate the endothelial-derived production of fibrotic components. Overall, our study demonstrates that the processing of myelin debris through the autophagy-lysosome pathway promotes inflammation and angiogenesis and may contribute to fibrotic scar formation.

Extracellular recombinant annexin II confers UVC-radiation resistance and increases the Bcl-xL to Bax protein ratios in human UVC-radiation-sensitive cells.

In this study, we found that refractoriness to ultraviolet (UVC) light-induced cell death was increased in UVC-radiation-sensitive cells derived from Cockayne syndrome patients when the cells were precultured in medium supplemented with recombinant annexin II (rANX II). In CS3BES cells, an immortal cell line derived from Cockayne syndrome patients, the rANX II supplementation-induced UVC-radiation resistance was suppressed by treatment with an anti-annexin II antibody and EGTA. The amount of biotinylated annexin II on the cell surface increased in the rANX II-supplemented cells but did not increase in the cells that were cotreated with rANX II and EGTA. The capacity to remove UVC-radiation-damaged DNA, (6-4) photoproducts and cyclobutane pyrimidine dimers, was the same in cells that were precultured with rANX II and in control cells that did not receive rANX II supplementation. The rANX II supplementation-induced UVC-radiation resistance was also observed in nucleotide excision repair-deficient cells and xeroderma pigmentosum group A-downregulated cells. The Bcl-xL to Bax protein ratios, an index of survival activity in cells exposed to lethal stresses, were increased in the cells that had been precultured in rANX II for 24 h prior to UVC irradiation. Treatment with a phosphatidylinositol 3-kinase inhibitor suppressed the increased UVC-radiation resistance and Bcl-xL to Bax ratios in the cells with rANX II supplementation. Furthermore, downregulation of Bcl-xL by siRNA transfection also suppressed the UVC-radiation resistance that was induced by rANX II supplementation. These results suggest that the increase in the Bcl-xL to Bax ratios may be associated with enhanced resistance to UVC-radiation-induced cell death.

Altered dendritic morphology of Purkinje cells in Dyt1 ΔGAG knock-in and purkinje cell-specific Dyt1 conditional knockout mice.

BACKGROUND: DYT1 early-onset generalized dystonia is a neurological movement disorder characterized by involuntary muscle contractions. It is caused by a trinucleotide deletion of a GAG (ΔGAG) in the DYT1 (TOR1A) gene encoding torsinA; the mouse homolog of this gene is Dyt1 (Tor1a). Although structural and functional alterations in the cerebellum have been reported in DYT1 dystonia, neuronal morphology has not been examined in vivo. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we examined the morphology of the cerebellum in Dyt1 ΔGAG knock-in (KI) mice. Golgi staining of the cerebellum revealed a reduction in the length of primary dendrites and a decrease in the number of spines on the distal dendrites of Purkinje cells. To determine if this phenomenon was cell autonomous and mediated by a loss of torsinA function in Purkinje cells, we created a knockout of the Dyt1 gene only in Purkinje cells of mice. We found the Purkinje-cell specific Dyt1 conditional knockout (Dyt1 pKO) mice have similar alterations in Purkinje cell morphology, with shortened primary dendrites and decreased spines on the distal dendrites. CONCLUSION/SIGNIFICANCE: These results suggest that the torsinA is important for the proper development of the cerebellum and a loss of this function in the Purkinje cells results in an alteration in dendritic structure.

The roles of HSP27 and annexin II in resistance to UVC-induced cell death: comparative studies of the human UVC-sensitive and -resistant cell lines RSa and APr-1.

We have reported that heat shock protein 27 (HSP27) and annexin II are involved in the protection of human cells against UVC-induced cell death. In this study we tried to confirm the combined roles of HSP27 and annexin II in cell death after UVC irradiation. In RSa cells with sensitivity to UVC, expression of annexin II decreased after UVC irradiation, but not in AP(r)-1 cells with increased resistance to UVC. HSP27 siRNA-transfected AP(r)-1 cells were sensitized to UVC lethality and showed decreased annexin II expression after UVC irradiation. In contrast, transfection of RSa cells with HSP27 cDNA increased their resistance to UVC lethality and caused increased annexin II expression. Furthermore, over-production of annexin II in RSa cells resulted in increased resistance to UVC lethality. This study indicates the involvement of cellular HSP27 expression in the UVC susceptibility of human cells, which occurs in association with regulation of annexin II expression.

Increase in the levels of chaperone proteins by exposure to beta-estradiol, bisphenol A and 4-methoxyphenol in human cells transfected with estrogen receptor alpha cDNA.

We examined changes in the levels of chaperone proteins to evaluate the toxic effects of environmental chemicals in human cells in vitro. Some chaperones are up-regulated by estrogenic chemicals, but the effect is not necessarily dependent on the receptor. Thus we also investigated whether a chemical-induced change in chaperone protein expression is human estrogen receptor (hER)-dependent or not, using cultured human cell lines transfected with hERalpha cDNA or an empty vector. In the hERalpha-expressed cells, the protein levels of the heat shock protein 27 (HSP27), the glucose-regulated protein 78 (GRP78/BiP), and GRP94 increased after exposure to beta-estradiol (E(2)) (from 10(-9)M to 10(-6)M) and bisphenol A (BPA) (from 10(-6)M to 10(-5)M). On the other hand, the increase was not observed in the cells without hERalpha expression. These results suggest that the E(2)- and BPA-induced increase in the protein levels were hERalpha dependent. We next examined the effect of four phenolic chemicals similar in structure to BPA, and found that among them, 4-methoxyphenol (from 10(-6)M to 10(-5)M) increased the levels of the chaperone proteins with hERalpha dependency. Thus the human cultured cells would be suitable for evaluating whether an increase in chaperone proteins occurs upon exposure to environmental chemicals and whether the effect is ER-dependent.

Annexin II, a novel HSP27-interacted protein, is involved in resistance to UVC-induced cell death in human APr-1 cells.

Heat shock protein 27 (HSP27) is implicated in diverse biologic functions as a molecular chaperone. We found that HSP27 is involved in the protection of human cells against UVC lethality. To elucidate the molecular mechanisms underlying UVC resistance, we searched for HSP27-interacted proteins related to resistance in UVC-resistant human cells, APr-1. Three candidates for HSP27-interacted proteins were found from cell lysates using an affinity column coupled with GST-fused HSP27 protein. Interaction between HSP27 and two candidates, annexin II and HSP70, was confirmed by immunoprecipitation analysis. After UVC irradiation, the amount of the complex of HSP27 and annexin II decreased in the postnuclear fraction, while it increased in the nuclear fraction. Cells transfected with annexin II-siRNA were more susceptible to UVC lethality. These results suggest that annexin II is a novel HSP27-interacted protein which is involved in UVC resistance in human cells, at least those tested here.