The Cstf2t Polyadenylation Gene Plays a Sex-Specific Role in Learning Behaviors in Mice

Polyadenylation is an essential mechanism for the processing of mRNA 30 ends. CstF-64 (the 64,000 Mr subunit of the cleavage stimulation factor; gene symbol Cstf2) is an RNAbinding protein that regulates mRNA polyadenylation site usage. We discovered a paralogous form of CstF-64 called τCstF-64 (Cstf2t). The Cstf2t gene is conserved in all eutherian mammals including mice and humans, but the τCstF-64 protein is expressed only in a subset of mammalian tissues, mostly testis and brain. Male mice that lack Cstf2t (Cstf2t-/- mice) experience disruption of spermatogenesis and are infertile, although female fertility is unaffected. However, a role for τCstF-64 in the brain has not yet been determined. Given the importance of RNA polyadenylation and splicing in neuronal gene expression, we chose to test the hypothesis that τCstF-64 is important for brain function. Male and female 185-day old wild type and Cstf2t-/- mice were examined for motor function, general activity, learning, and memory using rotarod, open field activity, 8-arm radial arm maze, and Morris water maze tasks. Male wild type and Cstf2t-/- mice did not show differences in learning and memory. However, female Cstf2t-/- mice showed significantly better retention of learned maze tasks than did female wild type mice. These results suggest that τCstf-64 is important in memory function in female mice. Interestingly, male Cstf2t-/- mice displayed less thigmotactic behavior than did wild type mice, suggesting that Cstf2t may play a role in anxiety in males. Taken together, our studies highlight the importance of mRNA processing in cognition and behavior as well as their established functions in reproduction

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

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

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