TLR4 in intestinal epithelial cells has been shown both inflammatory and homeostatic roles following binding of its cognate ligand lipopolysaccharide (LPS). TWEAK-Fn14 axis plays an important role in pathologies caused by excessive or abnormal inflammatory responses. This study aimed to evaluate potential cross-talk between TLR4 and TWEAK/Fn14 system in porcine small intestinal epithelial cells. Our in vivo results showed that, compared with the age-matched normal control piglets, increased expression of Fn14 in epithelium and decreased TWEAK expression in lamina propria were detected in the small intestinal of piglets stimulated with LPS. Consistent with this finding, treatment with LPS increased the expression of Fn14 and TLR4 while decreased TWEAK expression in porcine small intestinal epithelial cell lines SIEC02. Interestingly, modulating Fn14 activation using agonistic anti-Fn14 decreased TLR4-mediated TNF-α production by SIEC02. In addition, pretreatment of LPS-stimulated SIEC02 with recombinant TWEAK protein suppresses the expression of Fn14 and TNF-α and inhibits the negative impact of LPS on the tight junctional protein occludin expression. In conclusion, this study demonstrates that the TWEAK-independent Fn14 activation augments TLR4-mediated inflammatory responses in the intestine of piglets. Furthermore, the TWEAK-dependent suppression of Fn14 signaling may play a role in intestinal homeostasis.

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28Mutations of the X-linked methyl-CpG-binding protein 2 (MECP2) gene in humans are 29 responsible for most cases of Rett syndrome (RTT), an X-linked progressive neurological disorder. 30While genome-wide screens in clinical trials reveal several putative RTT-associated mutations on 31 MECP2, their causative relevance regarding the functional regulation of MeCP2 on the etiologic 32 sites at the protein level require more evidence. In this study, we demonstrate that MeCP2 is 33 dynamically modified by O-linked-β-N-acetylglucosamine (O-GlcNAc) at threonine 203 (T203), an 34 etiologic site in RTT patients. Disruption of the O-GlcNAcylation of MeCP2 specifically at T203 35 impairs dendrite development and spine maturation in cultured hippocampal neurons, and 36 disrupts neuronal migration, dendritic spine morphogenesis and dysfunction of synaptic 37 transmission in the developing and juvenile mouse cerebral cortex. Mechanistically, genetic 38 disruption of O-GlcNAcylation at T203 on MeCP2 decreases neuronal activity-induced induction 39 of Bdnf transcription. Our study highlights the critical role of MeCP2 T203 O-GlcNAcylation in 40 neural development and synaptic transmission potentially via BDNF. 41 42 Keywords: MeCP2; O-GlcNAcylation; dendrite development; synaptic transmission; BDNF 43 44 3 / 47Rett syndrome (RTT) is a severe X-linked neurodevelopmental disorder that preferentially 46 occurred in females, with an approximate incidence of 1:10,000 1, 2 . Almost 90% of RTT cases 47 are caused by methyl-CpG-binding protein 2 (MECP2) gene mutations such as missense, 48 nonsense, insertion, deletion, and splice-site variations 3, 4, 5 , and loss of MeCP2 is closely related 49 to the occurrence of RTT 1, 6, 7, 8 . MeCP2 activity in normal central nervous system (CNS) 50 development and function is controlled by both precise expression levels 9, 10 and post-51 translational modifications (PTMs). For example, neurons from Mecp2-null mutant mice have 52 smaller somas 11, 12 , decreased dendritic complexity 13, 14,15 and dysfunction of synaptic plasticity 53 16, 17, 18 . Gain-of-function MeCP2 by overexpression in transgenic mice or monkeys results in 54 progressive neurological and psychiatric dysfunctions 19,20 . These genetic studies suggest that 55 precise and dynamic expression of MeCP2 is critical to maintain normal brain development and 56 function. 57In addition to expression level, PTMs on MeCP2 such as phosphorylation have been 58 demonstrated as critical regulators of its role in dendritic growth, spine maturation, and activation 59 of calcium-dependent brain-derived neurotrophic factor (BDNF) gene expression, suggesting 60 that MeCP2 PTMs particularly impact neurodevelopment processes and activity-dependent 61 gene expression. 21, 22, 23,24, 25 . Recently, the novel PTM O-GlcNAcylation has emerged as a 62 potent regulator of neurogenesis and synaptic plasticity 26, 27,28 . O-GlcNAcylation is a highly 63 dynamic process 29, 30, 31 . O-GlcNAc transferase (OGT) catalyzes the addition of O-GlcNAc to 64 serine...