Altered neurite morphology and cholinergic function of induced pluripotent stem cell-derived neurons from a patient with Kleefstra syndrome and autism.

The aim of the present study was to establish an in vitro Kleefstra syndrome (KS) disease model using the human induced pluripotent stem cell (hiPSC) technology. Previously, an autism spectrum disorder (ASD) patient with Kleefstra syndrome (KS-ASD) carrying a deleterious premature termination codon mutation in the EHMT1 gene was identified. Patient specific hiPSCs generated from peripheral blood mononuclear cells of the KS-ASD patient were differentiated into post-mitotic cortical neurons. Lower levels of EHMT1 mRNA as well as protein expression were confirmed in these cells. Morphological analysis on neuronal cells differentiated from the KS-ASD patient-derived hiPSC clones showed significantly shorter neurites and reduced arborization compared to cells generated from healthy controls. Moreover, density of dendritic protrusions of neuronal cells derived from KS-ASD hiPSCs was lower than that of control cells. Synaptic connections and spontaneous neuronal activity measured by live cell calcium imaging could be detected after 5 weeks of differentiation, when KS-ASD cells exhibited higher sensitivity of calcium responses to acetylcholine stimulation indicating a lower nicotinic cholinergic tone at baseline condition in KS-ASD cells. In addition, gene expression profiling of differentiated neuronal cells from the KS-ASD patient revealed higher expression of proliferation-related genes and lower mRNA levels of genes involved in neuronal maturation and migration. Our data demonstrate anomalous neuronal morphology, functional activity and gene expression in KS-ASD patient-specific hiPSC-derived neuronal cultures, which offers an in vitro system that contributes to a better understanding of KS and potentially other neurodevelopmental disorders including ASD.

Selective pharmacological inhibition of the pacemaker channel isoforms (HCN1-4) as new possible therapeutical targets.

The pacemaker channel isoforms are encoded by the hyperpolarization-activated and cyclic nucleotide-gated (HCN) gene family and are responsible for diverse cellular functions including regulation of spontaneous activity in sino-atrial node cells and control of excitability in different types of neurons. Four channel isoforms exist (HCN1-HCN4). The hyperpolarization-activated cardiac pacemaker current (I(f)) has an important role in the generation of the diastolic depolarization in the sino-atrial node, while its neuronal equivalent (I(h)) is an important contributor to determination of resting membrane potential, and plays an important role in neuronal functions such as synaptic transmission, motor learning and generation of thalamic rhythms. Ivabradine is a novel, heart rate-lowering drug which inhibits the pacemaker (I(f)) current in the heart with high selectivity and with minimal effect on haemodynamic parameters. Ivabradine is beneficial in patients with chronic stable angina pectoris equally to beta receptor blocker and calcium channel antagonist drugs. There is increasing interest to apply this drug in other fields of cardiology such as heart failure, myocardial infarction, cardiac arrhyhtmias. Heart rate reduction might improve clinical outcomes in heart failure. HCN upregulation presumably contributes to increased (I(f)) and may play a role in ventricular and atrial arrhythmogenesis in heart failure. In the nervous system the HCN channels received attention in the research areas of neuropathic pain, epilepsy and understanding the mechanism of action of volatile anaesthetics. This article delineates that the pharmacological modulation of cardiac and neuronal HCN channels can serve current or future drug therapy and introduces some recently investigated HCN channel inhibitor compounds being potential candidates for development.