miR-142-5p Disrupts Neuronal Morphogenesis Underlying Porcine Hemagglutinating Encephalomyelitis Virus Infection by Targeting Ulk1

Li, Zi; Lan, Yungang; Zhao, Kui; Lv, Xiaoling; Ding, Ning; Lu, Huijun; Zhang, Jing; Yue, Huiqing; Shi, Junchao; Song, Deguang; Gao, Feng; He, Wenqi

In situ hybridization of brain tissue sections and primary cortical neurons showed that Ulk1 had a punctate distribution and was localized along the axon shaft and within growth cones ( Figure 6A) . Because the Ulk1 mRNAs were suppressed upon PHEV infection in neurons, it is possible that Ulk1 is involved in axonal growth and morphogenesis. To confirm a specific role for the Ulk1 proteins, we performed RNAi and overexpression analyses in primary cortical neurons. The siRNA construct directed against Ulk1 is outlined in the Table S2 , and its efficiency and specificity in the knockdown of target proteins was first confirmed by Western blot (Figure 6B) . To investigate the effect of overexpression of Ulk1, replication-deficient adenoviruses encoding Ulk1 (Ad5-Ulk1) were generated by homologous recombination, and it significantly increased the proportion of Ulk1 at a multiplicity of infection (MOI) of 100 compared with the untreated cells ( Figure 6C) . Antibody staining of primary cortical neurons revealed that disrupting Ulk1 in RNAiexpressing neurons mostly led to significantly shortened axon elongation and/or an abnormally large number of branched dendrites. In contrast, Ad5-Ulk1 infection resulted in excessive axon arborization and axon elongation, while the control neurons mostly grew in a normal mode with a main axon that extended over a long distance (Figure 6D) . Quantitative analyses of these phenotypes were described and summarized in a histogram, and the analyses indicated that Ulk1-RNAiexpressing neurons were less than approximately half the length of controls. Taken together, our data indicated that Ulk1 played a key role in neuronal morphogenesis and the dysfunctional Ulk1 disturbed axonal elongation in primary cortical neurons.