Regulation of Axon Guidance by Slit2 and Netrin-1 Signaling in the Lacrimal Gland of Aqp5 Knockout Mice

PURPOSE: Dry eye disease (DED) is multifactorial and associated with nerve abnormalities. We explored an Aquaporin 5 (AQP5)-deficiency-induced JunB activation mechanism, which causes abnormal lacrimal gland (LG) nerve distribution through Slit2 upregulation and Netrin-1 repression. METHODS: Aqp5 knockout (Aqp5(−)(/)(−)) and wild-type (Aqp5(+/+)) mice were studied. LGs were permeabilized and stained with neuronal class III β-tubulin, tyrosine hydroxylase (TH), vasoactive intestinal peptide (VIP), and calcitonin gene-related peptide (CGRP). Whole-mount images were acquired through tissue clearing and 3D fluorescence imaging. Mouse primary trigeminal ganglion (TG) neurons were treated with LG extracts and Netrin-1/Slit2 neutralizing antibody. Transcription factor (TF) prediction and chromatin immunoprecipitation-polymerase chain reaction (ChIP-PCR) experiments verified the JunB binding and regulatory effect on Netrin-1 and Slit2. RESULTS: Three-dimensional tissue and section immunofluorescence showed reduced LG nerves in Aqp5(−)(/)(−) mice, with sympathetic and sensory nerves significantly decreased. Netrin-1 was reduced and Slit2 increased in Aqp5(−)(/)(−) mice LGs. Aqp5(+/+) mice LG tissue extracts (TEs) promoted Aqp5(−)(/)(−) TG neurons axon growth, but Netrin-1 neutralizing antibody (NAb) could inhibit that promotion. Aqp5(−)(/)(−) mice LG TEs inhibited Aqp5(+/+) TG axon growth, but Slit2 NAb alleviated that inhibition. Furthermore, JunB, a Netrin-1 and Slit2 TF, could bind them and regulate their expression. SR11302, meanwhile, reversed the Netrin-1 and Slit2 shifts caused by AQP5 deficiency. CONCLUSIONS: AQP5 deficiency causes LG nerve abnormalities. Persistent JunB activation, the common denominator for Netrin-1 suppression and Slit2 induction, was found in Aqp5(−)(/)(−) mice LG epithelial cells. This affected sensory and sympathetic nerve fibers’ distribution in LGs. Our findings provide insights into preventing, reversing, and treating DED.