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

Neurons possess a soma, an axon and branched dendrites containing dendritic spines. To further define the physiological significance of PHEV infection in dendritic morphogenesis, we examined whether Ulk1 depression in mature 2-to 3-weekold neurons blocked spine formation. Without any treatment, normal spine formation occurs in vitro during the second and third weeks. Thus, we inoculated PHEV during this period and asked whether the virus inhibited endogenous Ulk1 and spine development. As shown in Figure 8A , control neurons showed robust spontaneous spine formation, but essentially no mature spines were observed in PHEV-infected neurons. And also, these PHEV-infected neurons exhibited stunted, bulbous spines, and numerous irregular membrane blebs, which gathered a large number of virus particles (Figure 8A) . The virus blocked spinal stretch, but there was a modest increase in the number, which might due to self-repair capability after injury. To further investigate whether Ulk1 deficits regulate dendritic spinal volume in PHEV-infected neurons, Ulk1 siRNA and Ad5-Ulk1 were introduced to assess sequence-specific inhibition and overexpression of endogenous Ulk1 function. Naturally, dendrite spines in Ulk1 siRNA-transfected neurons were morphologically immature and similar to the spines seen in PHEV-infected neurons, however, the phenotype was opposite in the overexpression group ( Figure 8B ). After transfection of miR-142-5p mimics, the neurons showed monstrous morphology that was similar to virus-infected neurons at the same stage ( Figure 8C ). Taken together, Ulk1 plays a key role in the branched dendrite outgrowth and dendritic spine formation, and Ulk1 down-regulation by miR-142-5p is responsible for the nerve damage caused by PHEV.