Castration mediated schwann cell dedifferentiation leads to slower nerve conduction, decreased neuritogenesis, and nitrergic neuron loss

Abstract

ABSTRACT Introduction/Objectives Low levels of androgens due to androgen deprivation therapy for prostate cancer treatment or due to aging lead to erectile dysfunction (ED). Castration, a preclinical model of low androgens, induces ED due to damage to pelvic nerves and a loss of erectogenic nitrergic nerves. Schwann cells (SC) release neurotrophic factors to promote pelvic nerve growth and survival. It is unknown how testosterone deficiency impacts SC function. This study determines how testosterone deficiency influences SC phenotype and trophic support in neuronal regeneration. We hypothesize testosterone deficiency will decrease myelinated pelvic SC populations, delay penile nerve conduction and reduce pelvic nitrergic neurons. Methods Male Sprague Dawley rats (12 wks) were divided into control (CON) and surgical castration (CAST) groups. All experiments were performed six weeks following castration. Dorsal penile nerves (DPN; 15-16 mm) were excised, placed in a sylgard dish containing oxygenated buffer and conduction velocity of myelinated fast fibers was assessed (n=8 per group). The major pelvic ganglia (MPG) were collected and dissociated into SC or neurons. Dissociated neurons were grown on laminin coated coverslips. To determine whether SCs could provide trophic support, we co-cultured neurons on top of confluent SCs. We assessed the following co-culture groups: CON SC + CON MPG, CON SC + CAST MPG, CAST SC + CON MPG (n=4 per group, cultured in triplicate). After 72 hours, all cultures were fixed and stained with beta tubulin class III, neuronal nitric oxide synthase (nNOS), and tyrosine hydroxylase (TH) to assess neurite length and branching, nitrergic and sympathetic neuron populations, respectively. Dissociated SC were characterized via qPCR for markers of all SC (Sox10), myelinated SC (Krox20), and dedifferentiated SC (glial fibrillary acidic protein; GFAP). Results CAST slows DPN myelinated fast fiber conduction velocity. In dissociated cultured MPG neurons, castration reduces both neurite length and number of branches. Interestingly, CAST MPG neurons co-cultured with CON SC restore neurite length and branching to same level as CON MPG neurons co-cultured with CON SC; however, CAST SC do not impact CON MPG neurite length. CAST MPG neurons co-cultured with CON SC and CON MPG neurons co-cultured with CAST SC both have less nitrergic neurons than CON SC + CON MPG neurons. CAST does not impact the number of TH neurons in single or co-cultured conditions. CAST reduces gene expression of Sox10, myelinated SC, and dedifferentiated SC. Conclusions CAST markedly impairs penile nerve conduction, reduces neurite outgrowth and the proportion of pro-erectile nitrergic neurons. Healthy SC are able to rescue neuritogenesis but not the survival of nitrergic neurons. Additionally, healthy MPG neurons plated on CAST SC have a reduced number of nitrergic neurons. We believe CAST SC are undergoing a phenotypic switch from a myelinated neurotrophic form into dedifferentiated SC promoting neuritogenesis and loss of nitrergic neurons. Preserving the myelinated SC phenotype may prevent impaired nerve damage and subsequent ED in patients with testosterone deficiency. Disclosure Work supported by industry: no.