τCstF-64 and CstF-64 are expressed in brains of adult mice.

<p>(A) Brain tissue extracts were separated by 10% SDS-PAGE and probed with antibodies that recognized τCstF-64 (Bethyl A301-487A, top panel), CstF-64 (3A7, middle panel), or β-tubulin (E7, bottom panel). Lanes were loaded with 10 μg total brain extract; lanes 1, female wild type; lanes 2, female <i>Cstf2t</i>^<i>-/-</i>; lanes 3, male wild type; lanes 4, male <i>Cstf2t</i>^<i>-/-</i>. (B) Testis (lanes 1, 2), brain (lanes 3, 4), and liver (lanes 5, 6) from wild type (lanes 1, 3, 5) or <i>Cstf2t</i>^<i>-/-</i> (lanes 2, 4, 6) male mice were separated by SDS-PAGE as in 1A. Extracts from primary rat neurons (lane 7) and rat C6 glioma cells (lane 8) were included for comparison. Blots were probed with anti-τCstF-64 (6A9, top panel), and then subsequently re-probed with anti-CstF-64 (3A7, bottom panel) antibodies. Apparent migrations of τCstF-64, CstF-64, and βCstF-64 are indicated by arrows.</p

Female wild type and <i>Cstf2t</i>^<i>-/-</i> mice display similar working and spatial memory as assessed by 8-arm radial arm maze (RAM).

<p>Male and female <i>Cstf2t</i>^-/- and wild type mice were run in a RAM once per day in five-day blocks with two days break between blocks for four weeks. <b>(A and D)</b> Comparison of <b>(A)</b> female and <b>(D)</b> male performance using “total # of entries,” an indicator of overall accuracy of learning and memory showed no significant differences between groups (2-way ANOVA (F (3, 1146) = 6.836; p<0.001)). A post-hoc Tukey’s multiple comparison test confirmed no differences between female and male <i>Cstf2t</i>^-/- and wild type groups, but indicated significant differences between female and male groups. <b>(B and E)</b> Comparison of <b>(B)</b> females and <b>(E)</b> males using the “total # of entries without error” metric, an estimate of memory span and, in turn, an estimate of working memory showed no significant differences between groups (2-way ANOVA (F (3, 1154) = 1.901; p = 0.13)). <b>(C and F)</b> Comparison of all groups examining “time to completion” of the maze task showed significant differences between groups in the “time to completion” metric (2-way ANOVA (F (3, 1151) = 27.99; p<0.0001)). A post-hoc Tukey’s multiple comparison test revealed no significant differences between <b>(C)</b> females, but revealed significant differences in <b>(F)</b> male wild type and <i>Cstf2t</i>^-/- mice (**p<0.001). All data are presented as mean ± SEM. ANOVA analyses were conducted using all groups.</p

Gross locomotor performances of <i>Cstf2t</i>^-/- and wild type mice are not significantly different.

<p>Female and male <i>Cstf2t</i>^-/- and wild type mice (185 ± 10 days of age) were run on a single mouse rotarod 3 times per day for 3 days to assess gross locomotor function. <b>(A and C)</b> Comparison of female and male mouse groups’ performance on rotarod indicated significant differences between groups (2-way ANOVA (F (3,528) = 27.78; p<0.0001). A post-hoc Tukey’s multiple comparison test indicated no significant differences between female wild type and <i>Cstf2t</i>^<i>-/-</i> and male wild type and <i>Cstf2t</i>^<i>-/-</i> mice. <b>(B and D)</b> Comparison of female and male mice indicated a group difference (1-way ANOVA (F (7, 123) = 23.36; p<0.0001. <b>(B)</b> A post-hoc Tukey’s multiple comparison of female groups indicated no significant differences between female <i>Cstf2t</i>^-/- and female wild type mouse weights <b>(D)</b> but did indicate a significant difference between male wild type and male <i>Cstf2t</i>^-/- mouse weights (** p<0.01). All data are presented as mean ± SEM. ANOVA analyses were conducted using all groups.</p

Male and female <i>Cstf2t</i>^<i>-/-</i> mice show reduced thigmotactic behavior, but males show it to a greater extent.

<p><b>(A and B)</b> Amount of time spent in thigmotaxis (activity while remaining near the wall), expressed as a percentage of total time. <b>(A)</b> Female <i>Cstf2t</i>^<i>-/-</i> mice showed no difference in thigmotaxis compared to female wild type mice. <b>(B)</b> Male <i>Cstf2t</i>^<i>-/-</i> mice showed significant reduction of thigmotaxis compared to male wild type mice (2-way ANOVA (F (3, 1328) = 59.38; p<0.0001). A post-hoc Tukey’s multiple comparison test indicated significant differences between male <i>Cstf2t</i>^<i>-/-</i> mice and male wild type mice (p<0.001). All data presented as mean ± SEM. **p<0.001. ANOVA analyses were conducted using all groups.</p

Templates for qPCR-based BER activity assay.

<p>Whole cell extracts were isolated from brain tissue and exposed to a template DNA containing a single nucleotide lesion (A) and control template (B). BER activity was calculated by comparing the ΔΔCt values (Ct is the number of cycles required for the fluorescent signal to cross the threshold) of the repaired and control templates.</p

OCM impairment is involved in ethanol-induced oxidative DNA damage and neuronal apoptosis effects in the PFC.

<p>The brain sections (PFC) of WT and <i>Mthfr+/−</i> mice exposed for 3 weeks or 4 days (acute) to the Lieber-DeCarli liquid diet with- or without ethanol (5%) were triple-labeled with NeuN (purple), TUNEL (green) and cleaved caspase-3 (red). Hoechst 33342 (blue) was used to identify all cell nuclei. Fluorescence was visualized by confocal microscopy. Scale bar = 20 µm. Note increased number of TUNEL/caspase-3-positive neurons in PFC of <i>Mthfr+/−</i> mice chronically exposed to ethanol, compared with corresponding WT mice (arrows). Also, note a higher density of TUNEL/caspase-3-positive neurons in PFC of chronically, compared with acutely exposed to ethanol WT mice (arrows).</p

PFC is more vulnerable to ethanol-induced oxidative stress than hippocampus.

<p>(A) oxo-8dG expression in neurons (neuronal marker MAP-2) was quantified in PFC and hippocampus (Hip) by stereological counting. Neither the number of neurons nor volumes of the brain structures were affected. Values are mean ± SEM; *p<0.01. (B) Oxo-8dG expression in neurons normalized to corresponding controls (Δ). Note significantly higher density of oxo-8dG -labeled neurons and Δ in PFC, compared with hippocampus of ethanol-exposed mice. (C) DNA repair activity in response to oxidative DNA damage (oxo-8dG) assessed by qPCR in whole cell extracts obtained from PFC and hippocampus (Hip) of control mice and mice exposed to acute or chronic ethanol. Values are means ± SEM; *p<0.05, **p<0.01. Note the response to oxidative DNA damage by PFC lysate is significantly stronger than those in the hippocampus.</p

OCM impairment is involved in ethanol impact on DNA repair in the PFC.

<p>(A) Blood Hcy levels in WT and <i>Mthfr+/−</i> mice following acute or chronic 3- week (3 w) or 5-week (5 w) ethanol exposure. Values are means ± SEM; *p<0.01; **p<0.001. Note chronic alcohol-induced increase in blood Hcy levels, compared with the acute exposure and the heightening this increase by MTHFR deficiency. (B) DNA repair activity in the PFC of WT and <i>Mthfr+/−</i> mice exposed to acute and chronic ethanol. Values are means ± SEM; *p<0.01; **p<0.005; ***p<0.002. Note a decrease in DNA repair activity in the PFC following 3-week (3 w) and even more so following 5 week (5 w) exposure, compared with acute alcohol exposure and a significant exaggeration of this decrease by MTHFR deficiency.</p

PFC is more vulnerable to ethanol-induced neuronal apoptosis than hippocampus.

<p>Brain sections double-labeled with TUNEL and MAP-2 in the PFC and hippocampus (Hip) and quantified by stereological counting; Values are means ± SEM; *p<0.01. Neither the number of MAP-2-positive cells (neurons) nor volumes of the brain structures were affected by chronic 3-week ethanol exposure. Note significantly higher density of TUNEL- positive neurons in PFC than in hippocampus of ethanol-exposed mice.</p