Journal: Molecular psychiatry

Article Title: Dysregulation of miR-34a Links Neuronal Development to Genetic Risk Factors for Bipolar Disorder

doi: 10.1038/mp.2014.176

Figure Lengend Snippet: A) Mean ± SEM miR-34a levels depicted as fold change relative to controls. N = 34 control and 29 BD cerebellar samples. **p<0.01 compared to control conditions, based on a two-tailed, one-sample t -test. B) Mean ± SEM miR-34a levels in cerebellum of controls (N = 34) and BD patients segregated into medicated (N = 19) and non-medicated (N = 10). **p<0.01 based on ANOVA with Tukey's Multiple Comparison Test. C) Schematic of the neuronal differentiation process from iPSCs to eight weeks differentiated neurons. D) Immunostaining of Nestin in hNPCs and MAP2 in eight weeks differentiated neurons, scale bar = 50 µm. E) Graphs showing relative miR-34a expression in hNPCs and differentiated neurons of a healthy control individual (light grey circles) and a BD patient (dark grey circles). Data are shown as fold change relative to miR-34a level at hNPC stage. Stars over data depict statistical significance based on a two-tailed t -test (*p<0.05, **p<0.01, *p<0.001). F) Schematic of induced neurons (iNs) generation. G) miR-34a expression in iNs from three control and five BD subjects. The graph shows mean ± SEM miR-34a expression relative to control iNs. miR-34a expression was normalized to U6 and RNU44 expression, which control for the total amount of RNA present in each sample. *p<0.05 compared to control conditions, based on a two-tailed, one-sample t -test.

Article Snippet: Expandable human neuronal progenitor cells (hNPCs) from one unaffected male (GM08330, Coriell Institute for Medical Research) and one BD male (GM05224, Coriell Institute for Medical Research) were generated from iPSCs (Madison et al. , manuscript under revision in Molecular Psychiatry) and cultured on poly-ornithine/laminin-coated six-well plates (BD Biosciences, San Jose, CA, USA) with neural expansion medium [70% Dulbecco’s Modified Eagle Medium (DMEM) (Invitrogen, Carlsbad, CA, USA), 30% Ham's F-12 (Mediatech, Manassas, VA, USA) supplemented with B-27 (Invitrogen), 20 ng/ml each EGF (Sigma Sigma-Aldrich, St Louis, MO, USA) and bFGF (R&D Systems, Minneapolis, MN, USA)].

Techniques: Control, Two Tailed Test, Comparison, Immunostaining, Expressing

Journal: Molecular psychiatry

Article Title: Dysregulation of miR-34a Links Neuronal Development to Genetic Risk Factors for Bipolar Disorder

doi: 10.1038/mp.2014.176

Figure Lengend Snippet: A) hNPCs were transduced with either miR-Ctrl or miR-34a lentiviral constructs and differentiated for eight weeks. Data show expression of miR-34a shown as fold change relative to the transcript level at the initial hNPC stage. B-C) Follow-up of the two validated miR-34a targets ANK3 and CACNB3 at transcriptional level in miR-Ctrl and miR-34a overexpressing hNPCs and differentiated neurons. Data are shown as fold change relative to the transcript of interest at hNPC stage and normalized to the transcript level in miR-Ctrl conditions. Data are mean ± SEM from three independent experiments. *p <0.05, **p<0.01, ***p<0.001 compared to miR-Ctrl treatment, based on a two-tailed, one-sample t -test. D) PSD95, Synaptophysin (SYP), Synapsin-1 (SYN1), ANK3 and CACNB3 Western blot results on hNPCs, four and eight weeks differentiated neurons overexpressing either miR-Ctrl or miR-34a (left panel) or two weeks differentiated control and BD neurons (right panel). β-actin was used as internal standard. E) Quantification of Western blot data in (D) by densitometric analysis. The bars show the mean ± SEM ratio of the protein of interest and β-actin signals, as evaluated by densitometric analysis of three independent biological replicates. *p <0.05, **p<0.01, ***p<0.001 compared to miR-Ctrl treatment, based on a two-way ANOVA using the Bonferroni correction as post hoc comparisons between two means. F) Image-based analysis of the effect of miR-34a overexpression on the morphology of hNPCs after three weeks of differentiation with sparse transfection with membrane-targeted GFP for two days, scale bar = 50 µm. G-H) Graph showing the number of branches in function of the soma distance in (G), three weeks differentiated neurons overexpressing miR-Ctrl (dark grey circles) and miR-34a (light grey circles) or in (H), two weeks differentiated neurons expressing anti-miR-scrambled control (dark grey circles) and anti-miR-34a (light grey circles). 12 days differentiated cells were transfected with anti-miR constructs for 2 days. Since both constructs co-expressed mCherry as a reporter gene, cells expressing the anti-miR constructs were immunostained for mCherry. For each condition, a total of 40 neurons were imaged and quantified using classical Sholl analysis. *p <0.05, **p<0.01, ***p<0.001 compared to miR-Ctrl treatment (G) or anti-miR-scrambled (H), based on a two-tailed t -test.

Article Snippet: Expandable human neuronal progenitor cells (hNPCs) from one unaffected male (GM08330, Coriell Institute for Medical Research) and one BD male (GM05224, Coriell Institute for Medical Research) were generated from iPSCs (Madison et al. , manuscript under revision in Molecular Psychiatry) and cultured on poly-ornithine/laminin-coated six-well plates (BD Biosciences, San Jose, CA, USA) with neural expansion medium [70% Dulbecco’s Modified Eagle Medium (DMEM) (Invitrogen, Carlsbad, CA, USA), 30% Ham's F-12 (Mediatech, Manassas, VA, USA) supplemented with B-27 (Invitrogen), 20 ng/ml each EGF (Sigma Sigma-Aldrich, St Louis, MO, USA) and bFGF (R&D Systems, Minneapolis, MN, USA)].

Techniques: Transduction, Construct, Expressing, Two Tailed Test, Western Blot, Control, Over Expression, Transfection, Membrane