A Potential Role For Dysregulated Synapse Apposition And Beta-3 Adrenergic Receptor Density In Neurogenic Detrusor Overactivity
Bryan S. Sack, MD1, Mary Piper, PhD2, Justin F. Cotellessa, BSc3, Claire Doyle, PhD1, Mehrnaz Gharaee-Kermani, DVM, PhD3, Amy Avery, MSc3, Fabliha Mahmood, BSc1, Jill A. Macoska, PhD3, Rosalyn M. Adam, PhD1.
1Boston Children's Hospital, Boston, MA, USA, 2Harvard T.H. Chan School of Public Health Bioinformatics Core, Boston, MA, USA, 3University of Massachusetts, Boston, MA, USA.
BACKGROUND: Neurogenic bladder and detrusor sphincter dyssynergia (DSD) can result in bladder outlet obstruction, high intravesical pressure, and detrusor overactivity (DO). Current therapies target the parasympathetic and sympathetic imbalance with type 3 muscarinic receptor antagonists and β3 adrenergic receptor agonists, respectively. Unfortunately, refractory urge incontinence and poor bladder compliance may lead to aggressive and potentially co-morbid surgical therapy. The symptoms of DO and urge incontinence are attributed to extensive fibroproliferative remodeling and neuronal dysregulation, but the specific molecular changes in the bladder and how they evolve over time are almost completely undefined. The aim of this study was to identify altered pathways and gene regulatory networks in a model of neurogenic bladder dysfunction. METHODS: Sprague-Dawley rats underwent mid-thoracic spinal cord transection to model neurogenic bladder and DSD. We harvested bladders and serum from injured and age-matched controls at 2, 8, and 16 wk after injury. Serum was assessed for markers of renal insufficiency and systemic morbidity. Collagen content of bladder tissues was quantified using Masson's trichrome staining. We isolated total RNA from full thickness bladder strips and cDNA library preparation was performed using a ribodepletion method. We performed RNA sequencing using an Illumina HiSeq 2500 generating 51 bp paired-end reads, to a depth of 28 million reads/sample with a >95% mapping rate and <1% ribosomal RNA mapping. rlog transformation of the RNA-seq data variance was performed using DESeq2. We performed Gene ontology (GO) term identification using gprofiler and clusterprofiler. Validation was performed on differentially expressed genes (DEGs) of interest in a separate cohort of animals using rtPCR. RESULTS: Collagen deposition in bladder tissue increased over time after SCI. Serum analysis revealed minimal systemic changes. Principal Component Analysis showed SCI and control groups clustered and separated well at each time point. 3025 DEGs were identified between control and SCI groups, with 207, 1355, and 2493 DEGs identified at the 2, 8, and 16-week time points, respectively. Significant GO terms included synapse organization, axon ensheathment, axon guidance, and neuromuscular junction function (Figure 1). DEGs of interest involved in bladder contractility (ADRB3, Chrm3), synapse organization (NLGN3, DCX), and neuronal signaling and differentiation (Gdf15, NTRK2) were validated using rtPCR (Table 1). CONCLUSIONS: We present the first deep sequencing and temporal analysis of the bladder transcriptome after SCI. Our findings suggest that decreased β3 adrenergic receptor levels may result in disproportionate parasympathetic activity leading to detrusor overactivity. Synaptic apposition is a coordinated event under the control of multiple synapse organizing proteins, many of which are downregulated in this model. Understanding synapse organization and β3 adrenergic receptor density could help identify mechanisms to better manage neurogenic bladder.
| Gene of Interest | 2wk | 8wk | 16wk |
| Beta-3 Adrenergic Receptor (ADRB3) | - | ⬇ | ⬇* |
| Type 3 muscarinic receptor (Chrm3) | - | - | - |
| Growth differentiation factor 15 (Gdf15) | - | ⬆* | - |
| TrkB Receptor (NTRK2) | ⬇ | ⬇ | ⬇ |
| Doublecortin (DCX) | ⬇ | ⬇* | ⬇* |
| Neuroligin 3 (NLGN3) | - | ⬇* | ⬇* |
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