Defects in early synaptic formation and neuronal function in Prader-Willi syndrome.

Prader-Willi syndrome (PWS), which is a complex epigenetic disorder caused by the deficiency of paternally expressed genes in chromosome 15q11-q13, is associated with several psychiatric dimensions, including autism spectrum disorder. We have previously reported that iPS cells derived from PWS patients exhibited aberrant differentiation and transcriptomic dysregulation in differentiated neural stem cells (NSCs) and neurons. Here, we identified SLITRK1 as a downregulated gene in NSCs differentiated from PWS patient iPS cells by RNA sequencing analysis. Because SLITRK1 is involved in synaptogenesis, we focused on the synaptic formation and function of neurons differentiated from PWS patient iPS cells and NDN or MAGEL2 single gene defect mutant iPS cells. Although ßIII tubulin expression levels in all the neurons were comparable to the level of differentiation in the control, pre- and postsynaptic markers were significantly lower in PWS and mutant neurons than in control neurons. PSD-95 puncta along ßIII tubulin neurites were also decreased. Membrane potential responses were measured while exposed to high K+ stimulation. The neuronal excitabilities in PWS and mutant neurons showed significantly lower intensity than that of control neurons. These functional defects in PWS neurons may reflect phenotypes of neurodevelopmental disorders in PWS.

Fatigue Effect on Minimal Toe Clearance and Toe Activity during Walking.

This study investigates the effects of fatigue on the process of walking in young adults using the developed clog-integrated sensor system. The developed sensor can simultaneously measure the forefoot activity (FA) and minimum toe clearance (MTC). The FA was evaluated through the change in the contact area captured by a camera using a method based on a light conductive plate. The MTC was derived from the distance between the bottom surface of the clog and ground obtained using a time of flight (TOF) sensor, and the clog posture was obtained using an acceleration sensor. The induced fatigue was achieved by walking on a treadmill at the fastest walking speed. We evaluated the FA and MTC before and after fatigue in both feet for 14 participants. The effects of fatigue manifested in either the FA or MTC of either foot when the results were evaluated by considering the participants individually, although individual variances in the effects of fatigue were observed. In the dominant foot, a significant increase in either the FA or MTC was observed in 13 of the 14 participants. The mean MTC in the dominant foot increased significantly (p = 0.038) when the results were evaluated by considering the participants as a group.

AKT-mediated enhanced aerobic glycolysis causes acquired radioresistance by human tumor cells.

BACKGROUND AND PURPOSE: Cellular radioresistance is a major impediment to effective radiotherapy. Here, we demonstrated that long-term exposure to fractionated radiation conferred acquired radioresistance to tumor cells due to AKT-mediated enhanced aerobic glycolysis. MATERIAL AND METHODS: Two human tumor cell lines with acquired radioresistance were established by long-term exposure to fractionated radiation with 0.5 Gy of X-rays. Glucose uptake was inhibited using 2-deoxy-D-glucose, a non-metabolizable glucose analog. Aerobic glycolysis was assessed by measuring lactate concentrations. Cells were then used for assays of ROS generation, survival, and cell death as assessed by annexin V staining. RESULTS: Enhanced aerobic glycolysis was shown by increased glucose transporter Glut1 expression and a high lactate production rate in acquired radioresistant cells compared with parental cells. Inhibiting the AKT pathway using the AKT inhibitor API-2 abrogated these phenomena. Moreover, we found that inhibiting glycolysis with 2-deoxy-D-glucose suppressed acquired tumor cell radioresistance. CONCLUSIONS: Long-term fractionated radiation confers acquired radioresistance to tumor cells by AKT-mediated alterations in their glucose metabolic pathway. Thus, tumor cell metabolic pathway is an attractive target to eliminate radioresistant cells and improve radiotherapy efficacy.

Cyclin D1 overexpression perturbs DNA replication and induces replication-associated DNA double-strand breaks in acquired radioresistant cells.

Fractionated radiotherapy (RT) is widely used in cancer treatment, because it preserves normal tissues. However, repopulation of radioresistant tumors during fractionated RT limits the efficacy of RT. We recently demonstrated that a moderate level of long-term fractionated radiation confers acquired radioresistance to tumor cells, which is caused by DNA-PK/AKT/GSK3ß-mediated cyclin D1 overexpression. The resulting cyclin D1 overexpression leads to forced progression of the cell cycle to S-phase, concomitant with induction of DNA double-strand breaks (DSBs). In this study, we investigated the molecular mechanisms underlying cyclin D1 overexpression-induced DSBs during DNA replication in acquired radioresistant cells. DNA fiber data demonstrated that replication forks progressed slowly in acquired radioresistant cells compared with corresponding parental cells in HepG2 and HeLa cell lines. Slowly progressing replication forks were also observed in HepG2 and HeLa cells that overexpressed a nondegradable cyclin D1 mutant. We also found that knockdown of Mus81 endonuclease, which is responsible for resolving aberrant replication forks, suppressed DSB formation in acquired radioresistant cells. Consequently, Mus81 created DSBs to remove aberrant replication forks in response to replication perturbation triggered by cyclin D1 overexpression. After treating cells with a specific inhibitor for DNA-PK or ATM, apoptosis rates increased in acquired radioresistant cells but not in parental cells by inhibiting the DNA damage response to cyclin D1-mediated DSBs. This suggested that these inhibitors might eradicate acquired radioresistant cells and improve fractionated RT outcomes.

Total synthesis of bicyclic depsipeptides spiruchostatins C and D and investigation of their histone deacetylase inhibitory and antiproliferative activities.

The bicyclic depsipeptide histone deacetylase (HDAC) inhibitors spiruchostatins C and D were synthesized for the first time in a highly convergent and unified manner. The method features the amide coupling of a D-leucine-D-cysteine- or D-valine-D-cysteine-containing segment with a D-alanine- or D-valine-containing segment to directly assemble the corresponding seco-acids, key precursors of macrolactonization. The HDAC inhibitory assay and cell-growth inhibition analysis of the synthesized depsipeptides determined the order of potency of spiruchostatins A-D in comparison with the clinically approved depsipeptide FK228 (romidepsin). Novel aspects of structure-activity relationships (SAR) were revealed.