Minimal data set

Minimal data set for 2017 PLOS ONE article "Serum copeptin and neuron specific enolase are markers of neonatal distress and long-term neurodevelopmental outcome"

Copeptin and NSE concentrations in serum samples over time (n = 75).

<p>(A, B) Copeptin levels decrease over time, with highest levels measured at 6 hours after birth. Copeptin concentrations are shown for individual neonates (A) as well as the median ±IQR concentration for each time point (B). Compared to the 6 hour time point, concentrations were significantly lower at all subsequent time points (p = 0.0448 at 6 vs. 12 hours, and p < 0.0001 at all other time points). (C, D) NSE levels are relatively stable until 24 h after birth, after which the levels decrease. Data is shown for individual neonates (C) and as the median ± IQR at each time point (D). Compared to the 6 hour time point, concentrations were significantly lower only at 168 hours after birth (p = 0.0013). The Kruskal-Wallis test with Dunn’s multiple comparisons test was used.</p

Clinical characteristics of the study groups.

<p>Clinical characteristics of the study groups.</p

Copeptin and NSE levels after birth in relation to two-year outcome.

<p>(A) Copeptin levels were significantly higher in neonates in the poor outcome group (n = 25) than in the favorable outcome group (n = 50) at 6 hours (p = 0.0068), 12 hours (p = 0.0050) and 48 hours (p = 0.0226) after birth. (B) NSE levels were significantly higher in the neonates in the poor outcome group compared to the favorable outcome group at all time points (p = 0.0009 at 6 hours, p = 0.0005 at 12 hours, p = 0.0005 at 24 hours, p = 0.0009 at 48 hours, and p = 0.0029 at 72 hours). (C-D) ROC curves of combined copeptin and NSE concentrations at 6 hours (C) and 12 hours (D) after birth in relation to two year neurodevelopmental outcome. Graphs A and B show the median ± IQR. The data for each time point were analyzed separately with the Mann-Whitney U-test, and the Holm-Sidak method was used to compute thresholds to ensure a family-wise error rate below 0.05.</p

Copeptin levels in relation to therapeutic hypothermia.

<p>Copeptin concentrations after birth were lower in the hypothermic neonates from the TOBY cohort (n = 11) compared to the normothermic neonates from the same cohort (n = 10) at 6 hours after birth (p = 0.0495). Graph shows the median ± IQR. The data for each time point was analyzed separately with the Mann-Whitney U-test, and the Holm-Sidak method was used to compute thresholds to ensure a family-wise error rate below 0.05.</p

Protein half-life of V5-CB1 and V5-CB2 splice isoforms differ.

<p><b>(A)</b> Protein stability of V5-CB1_SH3+ was determined by collecting the protein samples at 0, 1, 2, 4 and 8 hours in the presence of cyclohexamide (CHX) (0.1mM) and half-life relative to actin was plotted. The t_1/2 and R² values were determined (inset). <b>(B)</b> Protein half-life of CB2_SH3+ isoform. <b>(C)</b> Protein half-life of CB1_SH3- in HEK-293T cells. <b>(D)</b> Protein half-life of CB2_SH3- isoform in HEK-293T cells. CB1_SH3- shows the shortest half-life (1.8 hr), while CB2_SH3- has the longest half-life (7.294 hr). <b>(E)</b> Protein half-life of mCherry-CB1_SH3- in primary hippocampal neuron is similar to HEK-293T cells. <b>(F)</b> CB1 isoform C-terminal amino acid sequence with specific Ub lysine residues marked in red. <b>(G-J)</b> The predicted K491R/K492R ubiquitin residues in CB1 were mutated Lys/Arg and the protein stability of the mutants was determined. The ubiquitin site mutants have a longer half-life compared to the WT counterparts. <b>(I-J)</b> CBΔC_SH3+ and CBΔC_SH3- C-terminus deletion mutations show similar protein half-life.</p

V5-CB1 splice isoforms enhance eGFP-gephyrin clustering along proximal-distal axis.

<p><b>(A)</b> Cartoon of various CB isoforms and deletion mutations used in the current study. <b>(B-F)</b> 8+7 DIV neurons co-transfected with either V5-CB1_SH3+, V5-CB1_SH3-, V5-CB2_SH3+ or V5-CB2_SH3- isoform (blue) and eGFP-gephyrin (green) and stained for presynaptic marker VGAT (red). Alterations in the morphology of eGFP-gephyrin synaptic clusters was observed. <b>(B-F, lower panels)</b> zoom of dendritic proximal or distal dendritic segment showing eGFP-gephyrin clustering and vGAT co-localization. The localization of eGFP-gephyrin clusters with γ2 GABA_AR subunit was also observed. <b>(G-I)</b> Quantification of eGFP-gephyrin synaptic cluster density per 20 μm dendrite (DIV 8+7) at proximal and distal dendritic segments of neurons co-expressing eGFP-gephyrin and mCherry-CB1/2 isoforms. Scale bar 10μm and 5 μm. Statistical analysis for cluster density; One-way ANOVA, Bonferonni post-hoc test, p = 0.85; size analysis: Kruskal-Wallis non parametric test, Dunn's multiple comparison test p<0.0001.</p

CB1 and CB2 splice isoforms influence adult neurogenesis.

<p><b>(A-A”)</b> WT mice infected with eGFP or eGFP-CB1_SH3- retrovirus; <b>(B-B”)</b> WT mice infected with eGFP or eGFP-CB2_SH3- retrovirus and analyzed for cell migration at 14 dpi, 28dpi and 42 dpi. <b>(C-C”)</b> WT mice infected with eGFP or eGFP-CB1_SH3- retrovirus; <b>(D-D”)</b> WT mice infected with eGFP or eGFP-CB2_SH3- retrovirus and analyzed for dendrite maturation using Sholl analysis at 14 dpi, 28dpi and 42 dpi. <b>(E)</b> Morphology of adult newborn neurons infected with retrovirus expressing eGFP, eGFP-CB1_SH3- or eGFP-CB2_SH3-. <b>(F)</b> Total dendritic length of newborn neurons infected with eGFP, eGFP-CB1_SH3- or eGFP-CB2_SH3-. <b>(G)</b> Total number of terminal dendrites expressing eGFP or eGFP-CB2_SH3-. Kolmogorov-Smirnov (A-A”, B-B”), N = 38 to 54 cells/ group. Mann-Whitney test for AUC (C-C”, D-D”), N = 31–65 cells/group. Two-way ANOVA, Bonferroni post-hoc test (F,G), N = 34–45 cells/ group.</p

Cdc42DN and CB1 Ub mutant behave like CB2_SH3- <i>in vivo</i>.

<p><b>(A-A”)</b> Adult newborn neurons expressing eGFP-Cdc42 (DN) do not show any migration defects at 14 dpi, 28 dpi and 42 dpi. <b>(B-B”)</b> Adult newborn neurons expressing eGFP-Cdc42 (DN) show defect in dendrite maturation at 14 dpi, 28 dpi and 42 dpi. <b>(C-C’)</b> Adult newborn neurons expressing eGFP-CB1_SH3- 2R mutant exhibit reduced dendritic complexity at 28 dpi, but not 42 dpi. <b>(D)</b> Morphology of adult newborn neuron expressing eGFP, eGFP-Cdc42 (DN) or eGFP-CB1_SH3- 2R mutant. <b>(E)</b> Quantification of total dendrite length showing reduced length in neurons expressing eGFP-CB1_SH3- 2R mutant at 28 dpi, but not 42 dpi. <b>(F)</b> The number of terminal dendrites is reduced in neurons expressing eGFP-CB1_SH3- 2R mutant at both 28 dpi and 42 dpi. Kolmogorov-Smirnov test (A-A”), N = 38–51 cells/group. Mann-Whitney t-test AUC (B-B”, C-C’), N = 12–41 cells/group. Two-way ANOVA, Bonferroni post-hoc test (E,F), N = 12–25 cells/group.</p

CB1 and CB2 sequester Cdc42 and influence actin dynamics.

<p><b>(A-C)</b> Morphology of HEK-293T cells transfected with eGFP-CB2ΔPH, eGFP-CB1_SH3- or eGFP-CB2_SH3- and stained for actin using phalloidin. <b>(D)</b> Quantification of filopodia in HEK-293T cells expressing eGFP, eGFP-CB1_SH3+, eGFP-CB1_SH3-, eGFP-CB2_SH3+ or eGFP-CB2_SH3- shows an increase in cells expressing CB isoforms. <b>(E-G)</b> HEK-293T cell morphology after transfection with eGFP, eGFP and VSVG-Cdc42 (CA) or VSVG-Cdc42 (DN). Arrow heads show accumulation of VSVG-Cdc42 (DN) (red) in filopodia. <b>(H-I)</b> HEK-293T cells co-transfected with VSVG-Cdc42 (CA) and eGFP-CB1_SH3- or eGFP-CB2_SH3-. Arrow heads show accumulation of VSVG-Cdc42 (CA) (red) in filopodia. <b>(H’-I’)</b> HEK-293T cell morphology after co-transfection with VSVG-Cdc42 (DN) and eGFP-CB1_SH3- or eGFP-CB2_SH3- showing enhanced filopodia formation. Arrow head show accumulation of VSVG-Cdc42 (DN) (red) in filopodia. <b>(J-J’)</b> HEK-293T cell morphology after co-transfection with mCherry-gephyrin and eGFP-CB1_SH3- or eGFP-CB2_SH3-. Arrow show membrane ruffling instead of filopodia. <b>(K)</b> Quantification of filopodia in HEK-293T cells upon co-expression of VSVG-Cdc42CA, VSVG-Cdc42DN or mCherry-gephyrin along with V5-CB isoforms.</p