Autism-associated miR-873 regulates ARID1B, SHANK3 and NRXN2 involved in neurodevelopment

Autism spectrum disorders (ASD) are highly heritable neurodevelopmental disorders with significant genetic heterogeneity. Noncoding microRNAs (miRNAs) are recognised as playing key roles in development of ASD albeit the function of these regulatory genes remains unclear. We previously conducted whole-exome sequencing of Australian families with ASD and identified four novel single nucleotide variations in mature miRNA sequences. A pull-down transcriptome analysis using transfected SH-SY5Y cells proposed a mechanistic model to examine changes in binding affinity associated with a unique mutation found in the conserved 'seed' region of miR-873-5p (rs777143952: T > A). Results suggested several ASD-risk genes were differentially targeted by wild-type and mutant miR-873 variants. In the current study, a dual-luciferase reporter assay confirmed miR-873 variants have a 20-30% inhibition/dysregulation effect on candidate autism risk genes ARID1B, SHANK3 and NRXN2 and also confirmed the affected expression with qPCR. In vitro mouse hippocampal neurons transfected with mutant miR-873 showed less morphological complexity and enhanced sodium currents and excitatory neurotransmission compared to cells transfected with wild-type miR-873. A second in vitro study showed CRISPR/Cas9 miR-873 disrupted SH-SY5Y neuroblastoma cells acquired a neuronal-like morphology and increased expression of ASD important genes ARID1B, SHANK3, ADNP2, ANK2 and CHD8. These results represent the first functional evidence that miR-873 regulates key neural genes involved in development and cell differentiation.We acknowledge the financial support from Australian National Health and Medical Research Council (APP1008125)

Comparative Study on the Therapeutic Potential of Neurally Differentiated Stem Cells in a Mouse Model of Multiple Sclerosis

Background: Transplantation of neural stem cells (NSCs) is a promising novel approach to the treatment of neuroinflammatory diseases such as multiple sclerosis (MS). NSCs can be derived from primary central nervous system (CNS) tissue or obtained by neural differentiation of embryonic stem (ES) cells, the latter having the advantage of readily providing an unlimited number of cells for therapeutic purposes. Using a mouse model of MS, we evaluated the therapeutic potential of NSCs derived from ES cells by two different neural differentiation protocols that utilized adherent culture conditions and compared their effect to primary NSCs derived from the subventricular zone (SVZ). Methodology/Principal Findings: The proliferation and secretion of pro-inflammatory cytokines by antigen-stimulated splenocytes was reduced in the presence of SVZ-NSCs, while ES cell-derived NSCs exerted differential immunosuppressive effects. Surprisingly, intravenously injected NSCs displayed no significant therapeutic impact on clinical and pathological disease outcomes in mice with experimental autoimmune encephalomyelitis (EAE) induced by recombinant myelin oligodendrocyte glycoprotein, independent of the cell source. Studies tracking the biodistribution of transplanted ES cellderived NSCs revealed that these cells were unable to traffic to the CNS or peripheral lymphoid tissues, consistent with the lack of cell surface homing molecules. Attenuation of peripheral immune responses could only be achieved through multiple high doses of NSCs administered intraperitoneally, which led to some neuroprotective effects within the CNS

β³-tripeptides act as sticky ends to self-assemble into a bioscaffold

Peptides comprised entirely of β3-amino acids, commonly referred to as β-foldamers, have been shown to self-assemble into a range of materials. Previously, β-foldamers have been functionalised via various side chain chemistries to introduce function to these materials without perturbation of the self-assembly motif. Here, we show that insertion of both rigid and flexible molecules into the backbone structure of the β-foldamer did not disturb the self-assembly, provided that the molecule is positioned between two β3-tripeptides. These hybrid β3-peptide flanked molecules self-assembled into a range of structures. α-Arginlyglycylaspartic acid (RGD), a commonly used cell attachment motif derived from fibronectin in the extracellular matrix, was incorporated into the peptide sequence in order to form a biomimetic scaffold that would support neuronal cell growth. The RGD-containing sequence formed the desired mesh-like scaffold but did not encourage neuronal growth, possibly due to over-stimulation with RGD. Mixing the RGD peptide with a β-foldamer without the RGD sequence produced a well-defined scaffold that successfully encouraged the growth of neurons and enabled neuronal electrical functionality. These results indicate that β3-tripeptides can form distinct self-assembly units separated by a linker and can form fibrous assemblies. The linkers within the peptide sequence can be composed of a bioactive α-peptide and tuned to provide a biocompatible scaffold

Diminished hERG K+ channel activity facilitates strong human labour contractions but is dysregulated in obese women

Human ether-a-go-go-related gene (hERG) potassium channels determine cardiac action potential and contraction duration. Human uterine contractions are underpinned by an action potential that also possesses an initial spike followed by prolonged depolarization. Here we show that hERG channel proteins (α-conducting and ß-inhibitory subunits) and hERG currents exist in isolated patch-clamped human myometrial cells. We show that hERG channel activity suppresses contraction amplitude and duration before labour, thereby facilitating quiescence. During established labour, expression of ß-inhibitory protein is markedly enhanced, resulting in reduced hERG activity that is associated with an increased duration of uterine action potentials and contractions. Thus, changes in hERG channel activity contribute to electrophysiological mechanisms that produce contractions during labour. We also demonstrate that this system fails in women with elevated BMI, who have enhanced hERG activity as a result of low ß-inhibitory protein expression, which likely contributes to the weak contractions and poor labour outcomes observed in many obese women necessitating caesarean delivery

Suppression of <i>in vitro</i> MOG-specific T-cell responses.

<p>(A) Fixed numbers of splenocytes from 2D2 mice stimulated with MOG_35–55 were cultured in the presence of varying numers of GS-N cells, 46C-NS cells or SVZ-NSCs (expressed as NSC: splenocyte ratio). Proliferative responses were measured by ³H-thymidine incorporation and expressed as the mean counts per minute (CPM). (B–G) Supernatants were collected from co-cultures after 48 hrs and the level of pro-inflammatory cytokines was quantified by cytometric bead array. Data represent the mean ± SEM (n = 4 mice). *P&lt;0.05, **P&lt;0.01, ∧P&lt;0.005, ^#P&lt;0.001 vs MOG_35–55 stimulated splenocytes unless otherwise indicated.</p

Characterization of 46C-NS cells.

<p>(A–C) Immunofluorescence staining showed 46C-NS cells expressed nestin, red (A) and 3CB2, red (B) but not GFAP, green (A) or MAP2, green (B). (<b>D–E</b>) Following neuronal or astrocytic differentiation, 46C-NS cells expressed βIII-Tubulin, red and MAP2, green (C) or GFAP (D), respectively.</p

Primer sequences used for gene expression analysis.

<p>Primer sequences used for gene expression analysis.</p

Differentiation potential of GS-N cells.

<p>(A–C) Immunofluorescence staining showed GS-N cells expressed nestin, red (A), A2B5, red (B) and βIII-Tubulin, red and MAP2, green (C). (D–E) Following neuronal or astrocytic differentiation, GS-N cells expressed MAP2, green and NeuN, red (D) or GFAP (E), respectively. (F–K) Electrophysiological assessment of GS-N cell-derived neurons. Neurons in high density (F) fired spontaneous action potentials (APs), which were blocked by tetrodotoxin (TTX) (G). The depolarization giving rise to the APs (H) was underpinned by an inward current (I). Sparsely growing neurons (J) did not display spontaneous activity but APs could be evoked by depolarising current steps (K).</p