Distinctive genomic signature of neural and intestinal organoids from familial Parkinson's disease patient-derived induced pluripotent stem cells.

AIMS: The leucine-rich repeat kinase 2 (LRRK2) G2019S mutation is the most common genetic cause of Parkinson's disease (PD). There is compelling evidence that PD is not only a brain disease but also a gastrointestinal disorder; nonetheless, its pathogenesis remains unclear. We aimed to develop human neural and intestinal tissue models of PD patients harbouring an LRRK2 mutation to understand the link between LRRK2 and PD pathology by investigating the gene expression signature. METHODS: We generated PD patient-specific induced pluripotent stem cells (iPSCs) carrying an LRRK2 G2019S mutation (LK2GS) and then differentiated into three-dimensional (3D) human neuroectodermal spheres (hNESs) and human intestinal organoids (hIOs). To unravel the gene and signalling networks associated with LK2GS, we analysed differentially expressed genes in the microarray data by functional clustering, gene ontology (GO) and pathway analyses. RESULTS: The expression profiles of LK2GS were distinct from those of wild-type controls in hNESs and hIOs. The most represented GO biological process in hNESs and hIOs was synaptic transmission, specifically synaptic vesicle trafficking, some defects of which are known to be related to PD. The results were further validated in four independent PD-specific hNESs and hIOs by microarray and qRT-PCR analysis. CONCLUSION: We provide the first evidence that LK2GS also causes significant changes in gene expression in the intestinal cells. These hNES and hIO models from the same genetic background of PD patients could be invaluable resources for understanding PD pathophysiology and for advancing the complexity of in vitro models with 3D expandable organoids.

Mitofusins deficiency elicits mitochondrial metabolic reprogramming to pluripotency.

Cell reprogramming technology has allowed the in vitro control of cell fate transition, thus allowing for the generation of highly desired cell types to recapitulate in vivo developmental processes and architectures. However, the precise molecular mechanisms underlying the reprogramming process remain to be defined. Here, we show that depleting p53 and p21, which are barriers to reprogramming, yields a high reprogramming efficiency. Deletion of these factors results in a distinct mitochondrial background with low expression of oxidative phosphorylation subunits and mitochondrial fusion proteins, including mitofusin 1 and 2 (Mfn1/2). Importantly, Mfn1/2 depletion reciprocally inhibits the p53-p21 pathway and promotes both the conversion of somatic cells to a pluripotent state and the maintenance of pluripotency. Mfn1/2 depletion facilitates the glycolytic metabolic transition through the activation of the Ras-Raf and hypoxia-inducible factor 1α (HIF1α) signaling at an early stage of reprogramming. HIF1α is required for increased glycolysis and reprogramming by Mfn1/2 depletion. Taken together, these results demonstrate that Mfn1/2 constitutes a new barrier to reprogramming, and that Mfn1/2 ablation facilitates the induction of pluripotency through the restructuring of mitochondrial dynamics and bioenergetics.

Modification of octamer binding transcriptional factor is related to H2B histone gene repression during dimethyl sulfoxide-dependent differentiation of HL-60 cells.

Transcriptional regulation of H2B histone gene during dimethyl sulfoxide (DMSO)-dependent differentiation of HL-60 cells has been investigated using DNase I footprinting and DNA mobility shift assay. The level of histone H2B mRNA showed a slight decline at 2 days and hardly detectable at 4 days after DMSO treatment. H2B histone mRNA was repressed in proportion to the concentration of DMSO. In DNase I footprinting analysis, one nuclear factor (octamer binding transcription factor, OTF) bound at -42 bp (octamer motif, ATTTGCAT) in undifferentiated HL-60 cells. The binding pattern of OTF was unchanged during DMSO-dependent differentiation. One protein complex (OTF) was detected by DNA mobility shift assay in undifferentiated HL-60 cells. The mobility of OTF was partially retarded during DMSO-dependent differentiation and the retardant OTF was not newly synthesized protein. These results suggest that the posttranslational modification of OTF may be responsible for the repression of H2B histone gene during DMSO-dependent differentiation of HL-60 cells.

Association of castration-dependent early induction of c-myc expression with a cell proliferation of the ventral prostate gland in rat.

The protooncogene c-myc is known to be associated with both cell proliferation and apoptosis. The possible cellular affects of castration on the ventral prostate gland of rat as well as the relationship to a castration induced c-myc expression were examined. Levels of c-myc mRNA in the ventral prostate gland peaked at 6 h (early induction) and 48 h (late induction) after castration, respectively. Castration-induced DNA fragmentation was not observed at an early induction of c-myc mRNA. DNA fragmentation appeared to be testosterone-dependent. On the other hand, cellular DNA synthesis measured by [3H]thymidine uptake in the ventral prostate gland was increased to maximum at 6 h after castration. These results suggest that an early induction of c-myc mRNA in ventral prostate gland after castration is closely associated with cell proliferation of the gland.

Protein kinase CKII interacts with and phosphorylates the SAG protein containing ring-H2 finger motif.

To investigate the biological function of CKII, we have identified proteins that interact with the subunits of CKII using the yeast two-hybrid system. Here we report that SAG, an antioxidant protein containing Ring-H2 finger motif, is a cellular partner associating with the beta subunit of CKII. SAG does not interact with the alpha subunit of CKII. Analysis of SAG deletion mutants indicates that the Ring-H2 motif of SAG is necessary and sufficient for its binding to the beta subunit of CKII. Recombinant SAG can be phosphorylated by CKII in vitro, providing evidence that the beta subunit mediates the interaction of CKII enzyme with substrate proteins. Overlay experiment shows that SAG and the beta subunit of CKII associate directly in vitro and that CKII-mediated phosphorylation of SAG does not affect the interaction between SAG and the beta subunit of CKII. Northern blot analysis indicates that both SAG and the beta subunit of CKII were relatively rich in human heart, liver, skeletal muscle, and pancreas, but were detected in only trace amounts in brain, placenta, and lung. Our present results suggest that CKII may play a role in the regulation of SAG function.

Reduced level of ATF is correlated with transcriptional repression of DNA topoisomerase II alpha gene during TPA-induced differentiation of HL-60 cells.

DNA topoisomerase II is a marker for the proliferation state of mammalian cells in culture, and the protein levels are markedly higher in exponentially growing cells than quiescent cells and can be downregulated by growth of the cells at high density and serum starvation. Correlation between ATF and TPA-repressed DNA topoisomerase II alpha (Topo II alpha) mRNA has been investigated during TPA-induced differentiation of HL-60 cells. Topo II alpha mRNA and unknotting activity were reduced at 24 hours in TPA-treated HL-60 cells. The level of Topo II alpha mRNA and the activity were gradually decreased in proportion to the concentration of TPA. Two DNA-protein complexes were formed by DNA mobility shift assay when ATF-binding site was incubated with nuclear extract prepared from TPA-free HL-60 cells, and the amount of ATF was vanished after TPA treatment. TPA-repressed Topo II alpha mRNA and ATF levels were partially restored after pretreatment of staurosporin. These results suggest that the reduced level of ATF may be important to the transcriptional repression of Topo II alpha gene during TPA-induced differentiation in HL-60 cells and related to protein kinase C signal pathway.

TATA element-binding protein is important to epidermal growth factor-dependent induction of H2B histone gene expression in primary hepatocytes from rat.

Epidermal growth factor (EGF) is a potent mitogen for rat hepatocytes and mammalian histone synthesis is functionally and temporally coupled to DNA replication. To gain an insight on the role of EGF in the regulation of H2B histone gene expression in primary hepatocyte cultures, the binding patterns of nuclear proteins to various elements in the H2B histone gene upstream region have been investigated. EGF induced H2B histone mRNA with maximal stimulation reached at 36 hours. The induction of H2B histone mRNA was dependent on the concentration of EGF and almost reduced by actinomycin-D pretreatment. In DNase I footprinting analysis, one nuclear factor (TATA element-binding protein, TBP) bound at -20 bp (TATA element) in either the absence or presence of EGF. One DNA-protein complex was formed by DNA mobility shift assay when TATA element was incubated with nuclear extract prepared from EGF-free hepatocytes, and the amount of TBP was increased after EGF treatment. These results suggest that TBP may be correlated with transcriptional regulation of H2B histone gene by EGF in primary hepatocytes.

Molecular cloning of the leuB genes from Mycobacterium bovis BCG and Mycobacterium tuberculosis.

A gene responsible for the biosynthesis of leucine has been cloned by the complementation of the Escherichia coli leuB6 auxotroph mutant after transformation with the Mycobacterium bovis BCG genomic DNA library, which was constructed by ligating the partially digested BCG DNA with Sau3A1 into the pUC19 digested with BamHI. Sequencing of the leuB gene of BCG revealed an ORF (open reading frame) of 1.011 bp encoding isopropylmalate dehydrogenase with a calculated molecular weight of 42 kDa. The leuB gene of Mycobacterium tuberculosis isolated from Korean tuberculosis patient is shown to be identical to that of BCG except one bp.