(12) Do Long Non-Coding RNAs EVF1/2 Function in Interneuron Specification in Hypoxic-Ischemic Encephalopathy?

Background: Children born prematurely who experience brain injury around the time of birth are at significant risk for neurodevelopmental disorders including autism spectrum disorder (ASD) (incidence of 4–6%). Reduced interneurons (IN) in the cortex are a suspected neural basis for ASD. Transcription factors Dlx5 and Dlx6 regulate proper IN differentiation and interregional circuit formation. Previously, our lab showed that Evf (Erwinia virulence factor), with two isoforms Evf1 and Evf2, encodes a long non-coding RNA that represses expression of Dlx6 through antagonizing the Dlx5/6 ultraconserved enhancer (Dlx5/6UCE) by multiple mechanisms including inhibition of chromatin remodeling. Notably, INs are decreased in the hippocampi of Evf2 mutants.

Objectives: To test whether Evf1/2 deletion rescues interneurons and behavioral function following perinatal hypoxic-ischemic brain injury.

Design/Methods: Evf2TS/TS (null), Evf1/2TS/TS (null), and Evf1/2+/+ wildtype (WT) mice were used. Some mice were subjected to hypoxic-ischemic brain injury (HI) at postnatal day (P) 7 using the Vannucci model (right common artery ligation and transection followed by 8% oxygen for 60 minutes at 37°C) or sham surgery with normoxia (Sham). Ultrasonic vocalization assays (USV) were conducted at P11 to assess communication. Cylinder rearing tests were conducted at P17, P35 and P56 to assess spontaneous forelimb use. Immunofluorescent staining was performed using antibodies to VIP, CLB, SOM, PV, CLR, NPY, GAD. Cell counts were assessed at medial prefrontal cortex (mPFC).

Results: Evf2 deletion increased expression of VIP+, CLB+, and SOM+ interneurons (INs) and decreased PV+ INs in mPFC of uninjured Evf2TS/TS compared to WT (n=6 WT and 6 null, p< 0.05). After HI, there was a trend towards decreased vocalization in Evf1/2 TS/TS compared to Evf1/2+/+ Sham, Evf1/2+/+ HI, and Evf1/2 TS/TS Sham. In uninjured mice, left forelimb use at P35 was transiently decreased in Evf1/2 TS/TS compared to Evf1/2+/+ (n=19 null, 10 WT, p=0.03). At P56, there was no difference in forelimb use between the 4 groups.

Conclusions: Evf1/2 regulates IN subtype differentiation. Although modulating Evf1/2 expression may be a tractable strategy for rescuing INs after HI, this was not suggested by behavioral results of our experiments. Experiments are ongoing to quantify changes in IN after HI in Evf 1/2 null and WT mice. USV at later timepoints is warranted to fully exclude no benefit of Evf1/2 deletion.