Generation of three induced pluripotent stem cell lines from a patient with KCNQ2 developmental and epileptic encephalopathy as a result of the pathogenic variant c.638C > T; p.Arg213Gln (NUIGi063-A, NUIGi063-B, NUIGi063-C) and 3 healthy controls (NUIGi064-A, NUIGi064-B, NUIGi064-C)

KCNQ2 encodes the potassium-gated voltage channel Kv7.2, responsible for the M-current, which contributes to neuronal resting membrane potential. Pathogenic variants in KCNQ2 cause early onset epilepsies, developmental and epileptic encephalopathies. In this study, we generated three iPSC lines from dermal fibroblasts of a 5 year-old female patient with the KCNQ2 c.638C > T (p.Arg213Gln) pathogenic heterozygous variant and three iPSC lines from a healthy sibling control. These iPSC lines were validated by confirming the targeted mutation, SNP karyotyping, STR analysis, pluripotent gene expression, differentiation capacity into three germ layers, and were free of transgene integration and Mycoplasma. </p

Inhibition of IRE1α RNase activity sensitizes patient-derived acute myeloid leukaemia cells to proteasome inhibitors

Despite improvements in prognostic stratification and optimization of therapeutic intervention in acute myeloid leukaemia (AML) patients, long-term survival is low. Clinical trials suggest proteasome inhibitors may be beneficial, but further interrogation of the molecular consequences of proteasome inhibition in AML is warranted to identify novel approaches that enhance their efficacy.1 In multiple myeloma (MM), resistance to proteasome inhibitors can occur upon activation of the unfolded protein response (UPR), a stress response pathway that can control cell fate.2 Inositol-requiring enzyme 1 alpha (IRE1α) is one of three stress sensors that mediates UPR signalling. IRE1α activity occurs via its RNase domain resulting in cleavage of a 26-nucleotide intron from X-Box Binding Protein 1 (XBP1) mRNA leading to formation of a transcription factor, XBP1s. XBP1s enhances cell survival by increasing transcription of genes associated with protein folding, endoplasmic reticulum-associated degradation (ERAD) and phospholipid synthesis. We demonstrate that an IRE1 RNase inhibitor (MKC8866), in combination with proteasome inhibitors, significantly decreases XBP1s levels and increases cell death in AML cell lines and patient-derived AML cells. In addition, this combination treatment can successfully target the CD34+CD38− population and reduce clonogenic ability.</p

Additional file 1: Figure S1. of Rapamycin regulates autophagy and cell adhesion in induced pluripotent stem cells

Showing that rapamycin induces LC3B expression in human fibroblasts. Three lines of human fibroblasts (F1, F2, F3) were treated without (–) or with (+) 100 nM rapamycin overnight. The protein lysates were run of 15 % SDS-PAGE and blotted with anti-LC3B and anti-GAPDH. Two bands of LC3B-I (16 kDa) and LC3B-II (14 kDa) were seen. Rapamycin is shown to induce LC3B-I expression after 24 hours of treatment. (TIF 172 kb

NRXN1α+/- is associated with increased excitability in ASD iPSC-derived neurons

Background: NRXN1 deletions are identified as one of major rare risk factors for autism spectrum disorder (ASD) and other neurodevelopmental disorders. ASD has 30% co-morbidity with epilepsy, and the latter is associated with excessive neuronal firing. NRXN1 encodes hundreds of presynaptic neuro-adhesion proteins categorized as NRXN1α/β/γ. Previous studies on cultured cells show that the short NRXN1β primarily exerts excitation effect, whereas the long NRXN1α which is more commonly deleted in patients involves in both excitation and inhibition. However, patient-derived models are essential for understanding functional consequences of NRXN1α deletions in human neurons. We recently derived induced pluripotent stem cells (iPSCs) from five controls and three ASD patients carrying NRXN1α+/- and showed increased calcium transients in patient neurons. Methods: In this study we investigated the electrophysiological properties of iPSC-derived cortical neurons in control and ASD patients carrying NRXN1α+/- using patch clamping. Whole genome RNA sequencing was carried out to further understand the potential underlying molecular mechanism. Results: NRXN1α+/- cortical neurons were shown to display larger sodium currents, higher AP amplitude and accelerated depolarization time. RNASeq analyses revealed transcriptomic changes with significant upregulation glutamatergic synapse and ion channels/transporter activity including voltage-gated potassium channels (GRIN1, GRIN3B, SLC17A6, CACNG3, CACNA1A, SHANK1), which are likely to couple with the increased excitability in NRXN1α+/- cortical neurons. Conclusions: Together with recent evidence of increased calcium transients, our results showed that human NRXN1α+/- isoform deletions altered neuronal excitability and non-synaptic function, and NRXN1α+/- patient iPSCs may be used as an ASD model for therapeutic development with calcium transients and excitability as readouts.</p