Hippocampal neurons are inhibited by CSPGs, and rescued by GPX3 overexpression.

<p>Hippocampal neurons transfected with active genes were plated on CSPG or laminin substrates. <b><i>A</i></b>, CSPGs (white) strongly inhibited hippocampal growth compared to laminin (black) (p<0.0001, Mann Whitney U Test) in three independent experiments (triangle, square, and circle markers). Horizontal bar indicates the average neurite total length on CSPGs and laminin, 15.5 µm and 60.9 µm respectively. <b><i>B,C</i></b> Mean Z-Scores of transfected neurons with standard deviations, centered on the negative control, mCherry. Asterisks indicate significant effects (*, **, ***, p<0.05, 0.01, 0.001 Tukey-Kramer) compared to the mCherry control when analyzed with ANOVA (p<0.001). <b><i>D, E</i></b>, Representative images of hippocampal neurons growing on CSPGs transfected with GPX3 (<b>D</b>) or control mCherry (<b>E</b>). Scale bar 100 µm.</p

Active growth genes in cerebellar neurons and their expression pattern.

<p><b><i>A, B</i></b>, Phenotypic results of overexpression in cerebellar granule neurons (CGNs) confirmed by four replicates following the primary screen. Bars represent normalized values, centered on the neutral control, mCherry. Three parameters: number of branches (gray), neurite total length (white) and primary neurite count (black), are reported for transfected neurons (GFP positive). Significant results are indicated with asterisks (ANOVA). Genes had effects on CSPG (<b>A</b>), laminin (<b>B</b>), or both substrates. mCherry transfection with Gö6976, the positive control, is plotted on the far right. <b><i>C–F</i></b>, Representative images of CGNs transfected with the gene RBMX grown on CSPG (<b>C</b>) or laminin (<b>D</b>) substrates, or the peroxidase GPX3 grown on CSPGs (E) or laminin (F). <b><i>G–J</i></b>, Adult brain expression of four clones with significant phenotypic changes. Data were analyzed from the Allen Brain Atlas (<a href="http://www.brain-map.org" target="_blank">www.brain-map.org</a>) to determine the expression patterns and intensities of the active genes. <i>In-situ</i> hybridization demonstrated little mRNA expression for GPX3 (<b>G</b>), and RMBX (<b>H</b>), each of which promotes neurite growth. EIF2B5 (<b>I</b>), which also promoted growth had some expression throughout the brain, especially in CA1 and the dentate gyrus. Two of the inhibitory genes, SMARCAL1 (not shown) and DUS3L (<b>J</b>) had strong expression in the granule layer of the cerebellum. Expression intensity legend on the far right. Scale bar in <b><i>C–E</i></b> 200 µm. Scale bar in <b><i>G–J</i></b> 1 mm.</p

Significant morphological changes after peripheral gene expression in CGNs.

<p><b><i>A</i></b>, Vector grid of 154 genes from the screen, measuring neurite initiation on CSPG (vector points upward if growth was increased, downward if decreased) and laminin (vector points rightward if increased, leftward if decreased). Scale is the same for all genes and is indicated at the bottom corners (H6PD, DCTN2) and at the top right for the positive control Gö6976 (reported in Z-scores). White arrows: genes that significantly perturbed neurite initiation on both substrates, gray arrows significantly changed only on laminin, and black on CSPGs. Black dashed line separates CSPG effects between increase and decrease while gray dashed lines separates laminin effects. <b><i>B–E</i></b>, representative images of neurons growing on CSPG (upper panels) and laminin substrates (lower panels). Neurons expressing the gene WD repeat domain 33 (WDR33) had increased neurite initiation when grown on CSPGs but decreased neurite initiation when grown on laminin (<b>B</b>), while DUS3L “dihydrouridine synthase 3-like” acted as the strongest inhibitor of neurite initiation on both substrates (<b>C</b>). SEPT8 “Septin 8” increased neurite initiation on both substrates (<b>D</b>), and dynactin 2 (DCTN2) potentiated neurite initiation on laminin but not on CSPGs (<b>E</b>). Scale bar 200 µm.</p

GPX3 and GPX7 significantly increase neurite length of postnatal CGNs plated on the inhibitory MAG substrate.

<p>Postnatal day 8 rat CGNs were co-transfected with the pmaxGFP plasmid (green) and the pCMVSPORT6 plasmid expressing either GPX3, GPX7, OGFLR1 or the control gene mCherry and plated onto a feeder layer of CHO cells expressing a non-inhibitory construct (R2), or the CNS myelin component, MAG. <i>A</i>. CGNs growing on CHO-R2 transfected with GFP and mCherry. <i>B</i>. CHO-MAG strongly inhibited the neurite outgrowth of CGNs transfected with GFP and mCherry. <i>C</i>. CGN neurite outgrowth is partially rescued when transfected with GPX3. <i>D</i>. Data are plotted as mean +/− SEM of 8 experiments, (One-way ANOVA, Dunnett’s post hoc, *p<0.05, ***p<0.001). Red channel marks β-tubulin positive neurons, green channel represents GFP expression, transfected neurons therefore appear yellow.</p

Cerebellar granule neurons are robustly inhibited by CSPGs and partially rescued by Gö6976.

<p>Dissociated postnatal cerebellar granule neurons (CGNs) were challenged in an inhibitory assay. <b><i>A, B</i></b>, representative images of CGNs transfected with control plasmid pSport mCherry growing on CSPGs (<b>A</b>), and a permissive substrate, laminin (<b>B</b>). <b><i>C,D</i></b>, the addition of the PKC inhibitor Gö6976 partially relieved CSPG inhibition (<b>C</b>), and potentiated laminin growth (<b>D</b>). <b><i>E</i></b><i>,</i> Bar chart depicting ratios of seven parameters on CSPGs divided by laminin (with 95% confidence intervals). Growth on CSPGs led to large decreases in neurite length, the number of primary neurites, and the percentage of cells with neurites. Tubulin intensity and soma area were slightly increased. The neuron count was decreased on CSPGs, implying a deficiency in cell adhesion or survival. <b><i>F</i></b>, Percent increase by Gö6976 was plotted with 95% confidence intervals on CSPGs (open bars), and on laminin (solid bars). Gö6976 had a strong positive effect on neurons growing on CSPGs, especially for neurite length and the percentage of neurons growing neurites. Neuron count and viability was unchanged. Statistics with un-normalized treatment averages, Mann-Whitney Test, *p< = 0.05, **p< = 0.01, ***p< = 0.0001. Scale bar 100 µm.</p

Feedback provided by the Experimental Design Assistant (EDA).

<p>Feedback provided by the Experimental Design Assistant (EDA).</p

Example of an Experimental Design Assistant (EDA) diagram.

<p>EDA diagrams are composed of nodes and links to represent an entire experimental plan. Each node contains properties where specific details are captured (properties are not shown in this picture, but in the EDA they are accessible by clicking on the specific node). This particular example is a simple 2-group comparison. The grey nodes contain high-level information about the experiment, such as the null and alternative hypotheses, the effect of interest (via the experiment node), the experimental unit, or the animal characteristics. The blue and purple nodes represent the practical steps carried out in the laboratory, such as the allocation to groups (allocation node) and the group sizes and role in the experiment (group nodes), the treatments (via the intervention nodes), and the measurements taken (measurement nodes). The green and red nodes represent the analysis, the outcome measures, and the independent variables.</p