(168) Mechanisms by Which Silencing of Fidgetin-like 2 (FL2) During Cavernous Nerve Injury Accelerates Nerve Regeneration and Recovery of Erectile Function

Introduction Our previous work has demonstrated that Fidgetin-like 2 (FL2) is a microtubule-severing protein that negatively regulates both cell motility and axonal elongation in vitro. In a rat model of cavernous nerve (CN) transection, siRNA-mediated knockdown of FL2 (FL2-siRNA) at the time and site of injury promoted visible nerve regeneration across the nerve gap, enabling electrical stimulation of the CN to elicit erectile responses. Objective In the present study, to elucidate the molecular mechanisms underlying the recovery of erectile function following FL2 knockdown, we characterized the temporal transcriptomic changes in corporal tissue in a rat model of CN crush injury and correlated these changes with functional recovery of erectile responses. Methods Adult male rats underwent bilateral CN crush injury followed by local application of either FL2-siRNA or control-siRNA at the injury site. Erectile function was evaluated at 6- hours, 3-days, 1-week, 2-weeks, and 4-weeks post-injury using cavernosometry, the intracorporal pressure/blood pressure ratio (ICP/BP) in response to electrical CN stimulation. At each time point, penile tissue was collected for histological analysis or RNA extraction. Transcriptomic profiling was performed using RNA sequencing (RNA-seq) to characterize temporal changes in gene expression associated with treatment and functional recovery. Differential gene expression was analyzed using DESeq2 with FDR < 0.05. Results At 3- and 4- weeks post-injury, cavernosometry revealed that animals treated with FL2-siRNA exhibited significantly improved erectile function compared with those receiving control-siRNA. By 4 weeks, the ICP/BP response of FL2-siRNA–treated nerves approached that of naïve animals at low stimulation intensities. At higher levels of stimulation, an ICP/BP ratio of approximately 0.6 was achieved, corresponding to a clear erectile response. The improvement in erectile function was associated with distinct transcriptional changes in penile tissue. At 6-hours post-treatment, 168 genes were regulated by FL2-siRNA, whereas by 2-weeks this number had increased 18-fold to 3,173 genes. In contrast, control-siRNA treatment led to differential expression of 653 genes at 6 hours and 1,017 genes at 2 weeks (a 1.6-fold increase). Among the significantly enriched ontological categories regulated by FL2-siRNA were genes involved in neurogenesis and synaptic transmission, consistent with the re-establishment of neuronal signaling. Histological analysis further supported enhanced reinnervation in FL2-siRNA–treated animals. Four weeks after treatment, penile tissue from FL2-siRNA–treated animals exhibited significantly higher levels of nNOS compared with controls, indicating increased reinnervation of the penile shaft by regenerated nitrergic neurons. Conclusions Local FL2 knockdown following CN injury effectively induces nerve regeneration and reinnervation of erectile tissue, promoting the recovery of erectile function following CN injury. Our data suggests strategies that have translational potential in treating the erectile dysfunction that occurs following nerve injury associated with radical prostatectomy. These findings may have broader implications beyond sexual medicine, offering new insight into mechanisms that promote repair following peripheral or central nervous system injuries, such as sciatic nerve or spinal cord damage. Disclosure Any of the authors act as a consultant, employee or shareholder of an industry for: Microcures, Inc.