Whole-brain in vivo base editing reverses behavioral changes in Mef2c-mutant mice.

Whole-brain genome editing to correct single-base mutations and reduce or reverse behavioral changes in animal models of autism spectrum disorder (ASD) has not yet been achieved. We developed an apolipoprotein B messenger RNA-editing enzyme, catalytic polypeptide-embedded cytosine base editor (AeCBE) system for converting C·G to T·A base pairs. We demonstrate its effectiveness by targeting AeCBE to an ASD-associated mutation of the MEF2C gene (c.104T>C, p.L35P) in vivo in mice. We first constructed Mef2cL35P heterozygous mice. Male heterozygous mice exhibited hyperactivity, repetitive behavior and social abnormalities. We then programmed AeCBE to edit the mutated C·G base pairs of Mef2c in the mouse brain through the intravenous injection of blood-brain barrier-crossing adeno-associated virus. This treatment successfully restored Mef2c protein levels in several brain regions and reversed the behavioral abnormalities in Mef2c-mutant mice. Our work presents an in vivo base-editing paradigm that could potentially correct single-base genetic mutations in the brain.

Distinct recruitment dynamics of chandelier cells and basket cells by thalamocortical inputs.

Diverse types of GABAergic interneurons tend to specialize in their inhibitory control of various aspects of cortical circuit operations. Among the most distinctive interneuron types, chandelier cells (i.e., axo-axonic cells) are a bona fide cell type that specifically innervates pyramidal cells at the axon initial segment, the site of action potential initiation. Chandelier cells have been speculated to exert ultimate inhibitory control over pyramidal cell spiking. Thus, chandelier cells appear to share multiple similarities with basket cells, not only in firing pattern (fast spiking) and molecular components, but also in potentially perisomatic inhibitory control. Unlike basket cells, however, synaptic recruitment of chandelier cells is little known yet. Here, we examined the mediodorsal thalamocortical input to both chandelier cells and basket cells in medial prefrontal cortex, through combining mouse genetic, optogenetic and electrophysiological approaches. We demonstrated that this thalamocortical input produced initially weak, but facilitated synaptic responses at chandelier cells, which enabled chandelier cells to spike persistently. In contrast, this thalamocortical input evoked initially strong, but rapidly depressed synaptic responses at basket cells, and basket cells only fired at the initiation of input. Overall, the distinct synaptic recruitment dynamics further underscores the differences between chandelier cells and basket cells, suggesting that these two types of fast spiking interneurons play different roles in cortical circuit processing and physiological operation.

Target deletion of the cytoskeleton-associated protein palladin does not impair neurite outgrowth in mice.

Palladin is an actin cytoskeleton-associated protein which is crucial for cell morphogenesis and motility. Previous studies have shown that palladin is localized to the axonal growth cone in neurons and may play an important role in axonal extension. Previously, we have generated palladin knockout mice which display cranial neural tube closure defect and embryonic lethality before embryonic day 15.5 (E15.5). To further study the role of palladin in the developing nervous system, we examined the innervation of palladin-deficient mouse embryos since the 200 kd, 140 kd, 90-92 kd and 50 kd palladin isoforms were undetectable in the mutant mouse embryo brain. Contrary to the results of previous studies, we found no inhibition of the axonal extension in palladin-deficient mouse embryos. The cortical neurons derived from palladin-deficient mice also showed no significant difference in neurite outgrowth as compared with those from wild-type mice. Moreover, no difference was found in neurite outgrowth of neural stem cell derived-neurons between palladin-deficient mice and wild-type mice. In conclusion, these results suggest that palladin is dispensable for normal neurite outgrowth in mice.

[The effects of cysteines on the function of human glutathion S-transferase pi(GSTp) under cell oxidative stress].

Site-directed mutagenesis was used to generate three cysteine mutants of GSTp, C(47/101), C(14/47/101) and C(14/47/101/169). GSTp, C(47/101), C(14/47/101) and C(14/47/101/169) were transfected into 293 cells separately and GST activity was determined by using CDNB as substrate. Data showed that each cysteine mutant inhibited endogenous GST catalyzatic activity and had remarkable dominant negative effect. The expression vectors of wide type GSTp and its cysteine mutants were co-transfected with c-Jun, NF-kappaB, or p21 luciferase reporting vector, into 293 cells separately, luciferase activity showed that C(14/47/101) and C(14/47/101/169) can dramatically activate c-Jun and p21 transcriptional activity. Each cysteine mutant can increase endogenous p21 level, and also increased mortality rate of 293 cells when exposed to H2O2. These results suggest that cysteine residues of GSTp play an important role in protecting cells against oxitative stress.