Neurotrimin is an estrogen-inducible, uterine protein that mediates sympathetic axon repulsion

Sympathetic axon degeneration is a significant feature of many peripheral neuropathies. While much is known about axon sprouting and regeneration, molecular mechanisms underlying axon degeneration are poorly understood. To assess molecular mechanisms of axon degeneration, we investigated sympathetic remodeling in the female reproductive tract. Uterine smooth muscle contains abundant sympathetic innervation under low estrogen conditions, while increased estrogen levels result in reduced sympathetic innervation through axon degeneration. To identify candidate molecules involved in uterine sympathetic pruning we conducted microarray experiments on ovariectomized or estrogen treated rat myometrium. Data showed that myometrial neurotrimin (NTM) gene expression is upregulated by estrogen in an appropriate time-dependent manner. NTM is a member of the IgLON family of GPI-anchored neural cell adhesion molecules; while homophilic interactions promote cell adhesion and neurite outgrowth, heterophilic interactions mediate repulsive effects on sympathetic axons. Real time RT-PCR confirmed microarray data that estrogen upregulates NTM mRNA 4 fold at 6 h. Western blots showed that NTM protein is expressed in the myometrium and is upregulated under high estrogen conditions. To determine if NTM plays a functional role in estrogen-mediated sympathetic pruning, we employed sympathetic neuron/smooth muscle co-cultures in which NTM expression is impaired by siRNA. In cultures lacking estrogen, sympathetic neurons extended neurites that made physical contacts with uterine smooth muscle cells. When estrogen is added, sympathetic axon contact with the smooth muscle cells is reduced and total axon length is diminished. To achieve NTM downregulation, cultured smooth muscle cells were treated with NTM siRNA. NTM expression was effectively reduced by transient siRNA transfection of smooth muscle cells as confirmed by immunoblot, and this had no effect on neurite outgrowth in co-cultures lacking estrogen. However when neurons were co-cultured with smooth muscle cells transfected with NTM siRNA, estrogen treatment failed to inhibit sympathetic neurite outgrowth, indicating that in vitro NTM downregulation impairs estrogen's ability to induce sympathetic axon repulsion. These studies show that NTM plays an important role in determining sympathetic innervation density of a peripheral target organ, and that its expression is modulated by estrogen. Therefore, NTM appears to be an important factor in physiological neuroplasticity of the female reproductive tract. Supported by NIH NS053796.