NK and NKT cells in the pathogenesis of Hidradenitis suppurativa: Novel therapeutic strategy through targeting of CD2.

Hidradenitis suppurativa (HS) is a chronic debilitating inflammatory skin disease with poorly understood pathogenesis. Single-cell RNAseq analysis of HS lesional and healthy individual skins revealed that NKT and NK cell populations were greatly expanded in HS, and they expressed elevated CD2, an activation receptor. Immunohistochemistry analyses confirmed significantly expanded numbers of CD2+ cells distributed throughout HS lesional tissue, and many co-expressed the NK marker, CD56. While CD4+ T cells were expanded in HS, CD8 T cells were rare. CD20+ B cells in HS were localized within tertiary follicle like structures. Immunofluorescence microscopy showed that NK cells (CD2 + CD56 dim ) expressing perforin, granzymes A and B were enriched within the hyperplastic follicular epidermis and tunnels of HS and juxtaposed with apoptotic cells. In contrast, NKT cells (CD2 + CD3 + CD56 bright ) primarily expressed granzyme A and were associated with α-SMA expressing fibroblasts within the fibrotic regions of the hypodermis. Keratinocytes and fibroblasts expressed high levels of CD58 (CD2 ligand) and they interacted with CD2 expressing NKT and NK cells. The NKT/NK maturation and activating cytokines, IL-12, IL-15 and IL-18, were significantly elevated in HS. Inhibition of cognate CD2-CD58 interaction with blocking anti-CD2 mAb in HS skin organotypic cultures resulted in a profound reduction of the inflammatory gene signature and secretion of inflammatory cytokines and chemokines in the culture supernate. In summary, we show that a cellular network of heterogenous NKT and NK cell populations drives inflammation, tunnel formation and fibrosis in the pathogenesis of HS. Furthermore, CD2 blockade is a viable immunotherapeutic approach for the management of HS.

Epigenetic switch reshapes epithelial progenitor cell signatures and drives inflammatory pathogenesis in hidradenitis suppurativa.

Hidradenitis suppurativa (HS) is a complex inflammatory skin disease with undefined mechanistic underpinnings. Here, we investigated HS epithelial cells and demonstrated that HS basal progenitors modulate their lineage restriction and give rise to pathogenic keratinocyte clones, resulting in epidermal hyperproliferation and dysregulated inflammation in HS. When comparing to healthy epithelial stem/progenitor cells, in HS, we identified changes in gene signatures that revolve around the mitotic cell cycle, DNA damage response and repair, as well as cell-cell adhesion and chromatin remodeling. By reconstructing cell differentiation trajectory and CellChat modeling, we identified a keratinocyte population specific to HS. This population is marked by S100A7/8/9 and KRT6 family members, triggering IL1, IL10, and complement inflammatory cascades. These signals, along with HS-specific proinflammatory cytokines and chemokines, contribute to the recruitment of certain immune cells during the disease progression. Furthermore, we revealed a previously uncharacterized role of S100A8 in regulating the local chromatin environment of target loci in HS keratinocytes. Through the integration of genomic and epigenomic datasets, we identified genome-wide chromatin rewiring alongside the switch of transcription factors (TFs), which mediated HS transcriptional profiles. Importantly, we identified numerous clinically relevant inflammatory enhancers and their coordinated TFs in HS basal CD49fhigh cells. The disruption of the S100A enhancer using the CRISPR/Cas9-mediated approach or the pharmacological inhibition of the interferon regulatory transcription factor 3 (IRF3) efficiently reduced the production of HS-associated inflammatory regulators. Our study not only uncovers the plasticity of epidermal progenitor cells in HS but also elucidates the epigenetic mechanisms underlying HS pathogenesis.

Mechanism underlying follicular hyperproliferation and oncogenesis in hidradenitis suppurativa.

Hidradenitis suppurativa (HS) is a skin disorder that causes chronic painful inflammation and hyperproliferation, often with the comorbidity of invasive keratoacanthoma (KA). Our research, employing high-resolution immunofluorescence and data science approaches together with confirmatory molecular analysis, has identified that the 5'-cap-dependent protein translation regulatory complex eIF4F is a key factor in the development of HS and is responsible for regulating follicular hyperproliferation. Specifically, eIF4F translational targets, Cyclin D1 and c-MYC, orchestrate the development of HS-associated KA. Although eIF4F and p-eIF4E are contiguous throughout HS lesions, Cyclin D1 and c-MYC have unique spatial localization and functions. The keratin-filled crater of KA is formed by nuclear c-MYC-induced differentiation of epithelial cells, whereas the co-localization of c-MYC and Cyclin D1 provides oncogenic transformation by activating RAS, PI3K, and ERK pathways. In sum, we have revealed a novel mechanism underlying HS pathogenesis of follicular hyperproliferation and the development of HS-associated invasive KA.

Pulmonary pathogenesis in a murine model of inhaled arsenical exposure.

Arsenic trioxide (ATO), an inorganic arsenical, is a toxic environmental contaminant. It is also a widely used chemical with industrial and medicinal uses. Significant public health risk exists from its intentional or accidental exposure. The pulmonary pathology of acute high dose exposure is not well defined. We developed and characterized a murine model of a single inhaled exposure to ATO, which was evaluated 24 h post-exposure. ATO caused hypoxemia as demonstrated by arterial blood-gas measurements. ATO administration caused disruption of alveolar-capillary membrane as shown by increase in total protein and IgM in the bronchoalveolar lavage fluid (BALF) supernatant and an onset of pulmonary edema. BALF of ATO-exposed mice had increased HMGB1, a damage-associated molecular pattern (DAMP) molecule, and differential cell counts revealed increased neutrophils. BALF supernatant also showed an increase in protein levels of eotaxin/CCL-11 and MCP-3/CCL-7 and a reduction in IL-10, IL-19, IFN-γ, and IL-2. In the lung of ATO-exposed mice, increased protein levels of G-CSF, CXCL-5, and CCL-11 were noted. Increased mRNA levels of TNF-a, and CCL2 in ATO-challenged lungs further supported an inflammatory pathogenesis. Neutrophils were increased in the blood of ATO-exposed animals. Pulmonary function was also evaluated using flexiVent. Consistent with an acute lung injury phenotype, respiratory and lung elastance showed significant increase in ATO-exposed mice. PV loops showed a downward shift and a decrease in inspiratory capacity in the ATO mice. Flow-volume curves showed a decrease in FEV0.1 and FEF50. These results demonstrate that inhaled ATO leads to pulmonary damage and characteristic dysfunctions resembling ARDS in humans.

Cutaneous Exposure to Arsenicals Is Associated with Development of Constrictive Bronchiolitis in Mice.

Organoarsenicals, such as lewisite and related chloroarsine, diphenylchloroarsine (DPCA), are chemical warfare agents developed during World War I. Stockpiles in Eastern Europe remain a threat to humans. The well-documented effects of cutaneous exposure to these organoarsenicals include skin blisters, painful burns, and life-threatening conditions such as acute respiratory distress syndrome. In survivors, long-term effects such as the development of respiratory ailments are reported for the organoarsenical sulfur mustard; however, no long-term pulmonary effects are documented for lewisite and DPCA. No animal models exist to explore the relationship between skin exposure to vesicants and constrictive bronchiolitis. We developed and characterized a mouse model to study the long-term effects of cutaneous exposure on the lungs after exposure to a sublethal dose of organoarsenicals. We exposed mice to lewisite, DPCA, or a less toxic surrogate organoarsenic chemical, phenyl arsine oxide, on the skin. The surviving mice were followed for 20 weeks after skin exposure to arsenicals. Lung microcomputed tomography, lung function, and histology demonstrated increased airway resistance, increased thickness of the smooth muscle layer, increased collagen deposition in the subepithelium, and peribronchial lymphocyte infiltration in mice exposed to arsenical on skin.

Conformationally Defined Rexinoids for the Prevention of Inflammation and Nonmelanoma Skin Cancers.

Compound 1 is a potent rexinoid that is highly effective in cancer chemoprevention but elevates serum triglycerides. In an effort to separate the lipid toxicity from the anticancer activity of 1, we synthesized four new analogs of rexinoid 1, of which three rexinoids did not elevate serum triglycerides. Rexinoids 3 and 4 are twice as potent as rexinoid 1 in binding to Retinoid X receptor (RXR). All-trans retinoic acid (ATRA) plays a key role in maintaining skin homeostasis, and rexinoids 3-6 are highly effective in upregulating the genes responsible for the biosynthesis of ATRA. Inflammation plays a key role in skin cancer, and rexinoids 3 and 4 are highly effective in diminishing LPS-induced inflammation. Rexinoids 3 and 4 are highly effective in preventing UVB-induced nonmelanoma skin cancer (NMSC) without displaying any overt toxicities. Biophysical studies of rexinoids 3 and 5 bound to hRXRα-ligand binding domain (LBD) reveal important conformational and dynamical differences in the ligand binding domain.

Ex Vivo Culture Models of Hidradenitis Suppurativa for Defining Molecular Pathogenesis and Treatment Efficacy of Novel Drugs.

Hidradenitis suppurativa (HS) is a complex and debilitating inflammatory skin disease for which no effective treatment is available currently. This is partly because of the lack of adequate human or animal models for defining the pathobiology of the disease. Here, we describe the development of air-liquid (A-L) interface, liquid-submersion (L-S), and bioreactor (Bio) ex vivo skin culture models. All three ex vivo platforms were effective for culturing skin samples for up to 14 days. Tissue architecture and integrity remained intact for at least 3 days for healthy skin and 14 days for HS skin. Up to day 3, no significant differences were observed in % early apoptotic cells among all three platforms. However, late apoptotic/necrotic cell death was increased in HS skin at day 3 in A-L and Bio culture. These cultures efficiently support the growth of various cells populations, including keratinocytes and immune cells. Profiling inflammatory gene signatures in HS skin from these ex vivo cultures showed dynamic changes in expression at day 3 and day 14. All three culture platforms were necessary to represent the inflammatory gene status of HS skin at day 0, suggesting that not all gene clusters were identically altered in each culture method. Similarly, cytokine/chemokine profiling of the supernatants from vehicle- and drug-treated ex vivo HS cultures again showed a better prediction of drug efficacy against HS. Overall, development of these three culture systems collectively provides a powerful tool to uncover the pathobiology of HS progression and screen various drugs against HS.

Advances in molecular pathogenesis of hidradenitis suppurativa: Dysregulated keratins and ECM signaling.

Hidradenitis suppurativa (HS) is characterized by deep-seated, highly inflamed, and painful lumps/abscesses, fistulae, and sinus tracts that grow extensively deep in the dermis and are highly immunogenic in nature. In about one-third of the HS patients there is strong evidence for the role of γ-secretase mutations along with dysregulated Notch signaling. However, the contribution of dysregulated Notch signaling in HS pathogenesis in relation to hair follicle alterations and hyper-activation of the immune system remains undefined. A genome-wide association study (GWAS), proteomic data and functional investigations of identified sequence variants in HS pathology are not fully revealing. The disease initiation or progression may involve bacterial infection besides intrinsic functional defects in keratinocytes, which may be key to further exacerbate immune cell infiltration and cytokine production in and around the lesional tissue. The absence of a suitable animal model that could fully recapitulate the pathogenesis of HS is a major impediment for proper understanding the underlying mechanisms and development of effective treatments. The presence of extracellular matrix (ECM) degradation products along with dysregulation in keratinocytes and, dermal fibroblasts ultimately affect immune regulation and are various components of HS pathogenesis. Bacterial infection further exacerbates the complexity of the disease progression. While anti-TNFα therapy shows partial efficacy, treatment to cure HS is absent. Multiple clinical trials targeting various cytokines, complement C5a and ECM products are in progress. This review provides state-of-the-art information on these aspects with a focus on dysregulated keratinocyte and immune cells; and role of ECM, and Keratin functions in this regard.

Epigenetic regulation in the pathogenesis of non-melanoma skin cancer.

Understanding of cancer with the help of ever-expanding cutting edge technological tools and bioinformatics is revolutionizing modern cancer research by broadening the space of discovery window of various genomic and epigenomic processes. Genomics data integrated with multi-omics layering have advanced cancer research. Uncovering such layers of genetic mutations/modifications, epigenetic regulation and their role in the complex pathophysiology of cancer progression could lead to novel therapeutic interventions. Although a plethora of literature is available in public domain defining the role of various tumor driver gene mutations, understanding of epigenetic regulation of cancer is still emerging. This review focuses on epigenetic regulation association with the pathogenesis of non-melanoma skin cancer (NMSC). NMSC has higher prevalence in Caucasian populations compared to other races. Due to lack of proper reporting to cancer registries, the incidence rates for NMSC worldwide cannot be accurately estimated. However, this is the most common neoplasm in humans, and millions of new cases per year are reported in the United States alone. In organ transplant recipients, the incidence of NMSC particularly of squamous cell carcinoma (SCC) is very high and these SCCs frequently become metastatic and lethal. Understanding of solar ultraviolet (UV) light-induced damage and impaired DNA repair process leading to DNA mutations and nuclear instability provide an insight into the pathogenesis of metastatic neoplasm. This review discusses the recent advances in the field of epigenetics of NMSCs. Particularly, the role of DNA methylation, histone hyperacetylation and non-coding RNA such as long-chain noncoding (lnc) RNAs, circular RNAs and miRNA in the disease progression are summarized.

NETosis in the pathogenesis of acute lung injury following cutaneous chemical burns.

Despite the high morbidity and mortality among patients with extensive cutaneous burns in the intensive care unit due to the development of acute respiratory distress syndrome, effective therapeutics remain to be determined. This is primarily because the mechanisms leading to acute lung injury (ALI) in these patients remain unknown. We test the hypothesis that cutaneous chemical burns promote lung injury due to systemic activation of neutrophils, in particular, toxicity mediated by the deployment of neutrophil extracellular traps (NETs). We also demonstrate the potential benefit of a peptidyl arginine deiminase 4 (PAD4) inhibitor to prevent NETosis and to preserve microvascular endothelial barrier function, thus reducing the severity of ALI in mice. Our data demonstrated that phenylarsine oxide (PAO) treatment of neutrophils caused increased intracellular Ca2+-associated PAD4 activity. A dermal chemical burn by lewisite or PAO resulted in PAD4 activation, NETosis, and ALI. NETs disrupted the barrier function of endothelial cells in human lung microvascular endothelial cell spheroids. Citrullinated histone 3 alone caused ALI in mice. Pharmacologic or genetic abrogation of PAD4 inhibited lung injury following cutaneous chemical burns. Cutaneous burns by lewisite and PAO caused ALI by PAD4-mediated NETosis. PAD4 inhibitors may have potential as countermeasures to suppress detrimental lung injury after chemical burns.

In-silico Studies and Wet-Lab Validation of Camptothecin Derivatives for Anti-Cancer Activity Against Liver (HepG2) and Lung (A549) Cancer Cell Lines.

BACKGROUND: In the present study, we have explored the utility of QSAR modelling, in silico ADMET, docking, chemical semi-synthesis, and in vitro evaluation studies for the identification of active camptothecin (CPT) derivatives against cancer-targeting human liver (HepG2) and lung (A549) cancer cell lines. METHODS: Two QSAR models were developed as screenings tools using the multiple linear regression (MLR) method followed by ADMET and docking studies. The regression coefficient (r2) and cross-validation regression coefficients (rCV2T) of the QSAR model for the HepG2 cell line was 0.95 and 0.90, respectively, and for the A549 cell line, it was 0.93 and 0.81, respectively. RESULTS: In silico studies show that CPT derivatives (CPT-1 and CPT-6) possess drug-like properties. Docking performed on DNA Topoisomerase-I showed significant binding affinity. Finally, predicted active derivatives were chemically semi synthesized, spectroscopically characterized, and evaluated in-vitro for cytotoxic/anticancer activity against HepG2 and A549 cell lines. CONCLUSION: The experimental results are consistent with the predicted results. These findings may be of immense importance in the anticancer drug development from an inexpensive and widely available natural product, camptothecin.

Constitutively active HCN channels constrain detrusor excitability and modulate evoked contractions of human bladder.

OBJECTIVE: Expression of Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels is reported in bladder, but the functional role remains unsettled. Here, we immunolocalized the HCN1 and HCN4 subtype in human bladder and investigated their functional significance. METHODS: Bladder procured from ten organ donors was dissected into mucosa (containing urothelium and submucosa) and detrusor for double immunofluorescence of HCN1 and 4 subtypes with gap junction and neural proteins together with isometric tension recordings. Mucosa intact and denuded detrusor strips were stretched to a basal tension of 10 mN for eliciting either tetrodotoxin (TTX) resistant spontaneous, carbachol evoked contractions and TTX sensitive electrical field stimulated (EFS), pre and post-addition of HCN blocker, ZD7288 or the activator, Lamotrigine or the cholinesterase inhibitor, Neostigmine. RESULTS: Double immunofluorescence revealed immunolocalization of HCN1 and HCN4 subtype with calcitonin gene related peptide (CGRP), choline acetyl transferase and gap junction proteins in mucosa and detrusor. Removal of mucosa significantly raised the resting tension and the force of spontaneous contractions upon cumulative addition of ZD7288 in micromolar range relative to Lamotrigine treated strips (P<0.05). ZD7288 [10 nM] did not affect the contractile response evoked by EFS or carbachol, but the addition of ZD7288 [10 nM] in presence of Neostigmine [1 µM] significantly enhanced the atropine and TTX sensitive EFS evoked contractions of mucosa denuded strips. CONCLUSIONS: Overall, HCN channels immunolocalized in mucosa, smooth muscle, gap junctions and nerve fibers exert a tonic constraint on detrusor excitability, enable spatio-temporal integration of evoked contractions and constrain the release of neurotransmitters, respectively. In contrast to the pacemaker role in other organs, findings argue for a non-pacemaking role of HCN channels in human bladder.

Patched1 haploinsufficiency severely impacts intermediary metabolism in the skin of Ptch1+/-/ODC transgenic mice.

The study of dominantly heritable cancers has provided insights about tumor development. Gorlin syndrome (GS) is an autosomal dominant disorder wherein affected individuals develop multiple basal cell carcinomas (BCCs) of the skin. We developed a murine model of Ptch1 haploinsufficiency on an ornithine decarboxylase (ODC) transgenic background (Ptch1+/-/ODCt/C57BL/6) that is more sensitive to BCCs growth as compared with Ptch1+/+/ODCt/C57BL/6 littermates. Ptch1+/-/ODCt/C57BL/6 mice show an altered metabolic landscape in the phenotypically normal skin, including restricted glucose availability, restricted ribose/deoxyribose flow and NADPH production, an accumulation of α-ketoglutarate, aconitate, and citrate that is associated with reversal of the tricarboxylic acid cycle, coupled with increased ketogenic/lipogenic activity via acetyl-CoA, 3-hydroybutyrate, and cholesterol metabolites. Also apparent was an increased content/acetylation of amino-acids, glutamine and glutamate, in particular. Accordingly, metabolic alterations due to a single copy loss of Ptch1 in Ptch1+/-/ODCt/C57BL/6 heterozygous mice may provide insights about the cancer prone phenotype of BCCs in GS patients, including biomarkers/targets for early intervention.

Correction to: Differentiating neurons derived fromhuman umbilical cord blood stem cells work as a test system for developmental neurotoxicity.

The original version of this article unfortunately contained a mistake. The acknowledgment published was incomplete.

Drug Targets in Neurotrophin Signaling in the Central and Peripheral Nervous System.

Neurotrophins are a family of proteins that play an important role in the regulation of the growth, survival, and differentiation of neurons in the central and peripheral nervous system. Neurotrophins were earlier characterized by their role in early development, growth, maintenance, and the plasticity of the nervous system during development, but recent findings also indicate their complex role during normal physiology in both neuronal and non-neuronal tissues. Therefore, it is important to recognize a deficiency in the expression of neurotrophins, a major factor driving the debilitating features of several neurologic and psychiatric diseases/disorders. On the other hand, overexpression of neurotrophins is well known to play a critical role in pathogenesis of chronic pain and afferent sensitization, underlying conditions such as lower urinary tract symptoms (LUTS)/disorders and osteoarthritis. The existence of a redundant receptor system of high-and low-affinity receptors accounts for the diverse, often antagonistic, effects of neurotrophins in neurons and non-neuronal tissues in a spatial and temporal manner. In addition, studies looking at bladder dysfunction because of conditions such as spinal cord injury and diabetes mellitus have found alterations in the levels of these neurotrophins in the bladder, as well as in sensory afferent neurons, which further opens a new avenue for therapeutic targets. In this review, we will discuss the characteristics and roles of key neurotrophins and their involvement in the central and periphery nervous system in both normal and diseased conditions.

MicroRNAs as potential biomarkers to predict the risk of urinary retention following intradetrusor onabotulinumtoxin-A injection.

AIMS: MicroRNAs (miRs) control post-transcriptional gene expression, and this is relevant in understanding better chronic diseases and treatment outcomes. The role of miRs in the pathology and treatment outcomes of overactive bladder (OAB) is unknown. In this study, we assessed the differential expression of miRs in OAB patients responding with either normal or elevated post-void residual volumes (PVRs) ≥200 mL following intradetrusor injection of onabotulinumtoxin-A (onaBoNT-A). METHODS: Female OAB patients refractory to OAB drugs were consented for this study. Cystoscopic-guided punch bladder biopsy was obtained at the time of injection of onaBoNT-A 100 units. The expression of 13 miR species, selected for their known effect on neurotrophin expression and smooth muscle function, was measured. PVRs and urine nerve growth factor (NGF) levels were measured at baseline and at the follow-up visit. RESULTS: Fourteen patients with mean age of 66 years were consented. Of these patients, nine maintained PVRs <200 mL after onaBoNT-A injection to comprise the low PVR group. The other five patients with PVRs ≥200 mL comprised the high PVR group. The expression of miR221 and miR125b was upregulated by 11- and 2-fold, respectively, in patients who responded with low PVRs after onaBoNT-A (P < 0.05). Urine NGF levels at baseline were not different between the two groups. CONCLUSIONS: This study suggests that deficiency in the pretreatment expression of miR221 and miR125b may predispose OAB patients to high PVRs following intradetrusor onaBoNT-A. Additional studies are needed to better understand the role of miRs in OAB.

Molecular correlates in urine for the obesity and prostatic inflammation of BPH/LUTS patients.

PURPOSE: Benign prostatic hyperplasia (BPH) is strongly associated with obesity and prostatic tissue inflammation, but the molecular underpinning of this relationship is not known. Here, we examined the association between urine levels of chemokines/adipokines with histological markers of prostate inflammation, obesity, and lower urinary tract symptoms LUTS in BPH patients. METHODS: Frozen urine specimens from 207 BPH/LUTS patients enrolled in Nashville Men's Health Study were sent for blinded analysis of 11 analytes, namely sIL-1RA, CXC chemokines (CXCL-1, CXCL-8, CXCL-10), CC chemokines (CCL2, CCL3, CCL5), PDGF-BB, interleukins IL-6, IL-17, and sCD40L using Luminex™ xMAP® technology. After adjusting for age and medication use, the urine levels of analytes were correlated with the scales of obesity, prostate inflammation grade, extent, and markers of lymphocytic infiltration (CD3 and CD20) using linear regression. RESULTS: sIL-1RA levels were significantly raised with higher BMI, waist circumference and waist-hip ratio in BPH patients after correction for multiple testing (P = 0.02). Men with greater overall extent of inflammatory infiltrates and maximal CD3 infiltration were marginally associated with CXCL-10 (P = 0.054) and CCL5 (P = 0.054), respectively. CCL3 in 15 patients with moderate to severe grade inflammation was marginally associated with maximal CD20 infiltration (P = 0.09), whereas CCL3 was undetectable in men with mild prostate tissue inflammation. There was marginal association of sCD40L with AUA-SI scores (P = 0.07). CONCLUSIONS: Strong association of sIL-1RA in urine with greater body size supports it as a major molecular correlate of obesity in the urine of BPH patients. Increased urine levels of CXCL-10, CCL5, and CCL3 were marginally associated with the scores for prostate tissue inflammation and lymphocytic infiltration. Overall, elevated urinary chemokines support that BPH is a metabolic disorder and suggest a molecular link between BPH/LUTS and prostatic inflammation.

BDNF overexpression in the bladder induces neuronal changes to mediate bladder overactivity.

Elevated levels of brain-derived neurotrophic factor (BDNF) in urine of overactive bladder (OAB) patients support the association of BDNF with OAB symptoms, but the causality is not known. Here, we investigated the functionality of BDNF overexpression in rat bladder following bladder wall transfection of either BDNF or luciferase (luciferase) transgenes (10 µg). One week after transfection, BDNF overexpression in bladder tissue and elevation of urine BDNF levels were observed together with increased transcript of BDNF, its cognate receptors (TrkB and p75NTR), and downstream PLCγ isoforms in bladder. BDNF overexpression can induce the bladder overactivity (BO) phenotype which is demonstrated by the increased voiding pressure and reduced intercontractile interval during transurethral open cystometry under urethane anesthesia. A role for BDNF-mediated enhancement of prejunctional cholinergic transmission in BO is supported by the significant increase in the atropine- and neostigmine-sensitive component of nerve-evoked contractions and upregulation of choline acetyltransferase, vesicular acetylcholine transporter, and transporter Oct2 and -α1 receptors. In addition, higher expression of transient receptor channels (TRPV1 and TRPA1) and pannexin-1 channels in conjunction with elevation of ATP and neurotrophins in bladder and also in L6/S1 dorsal root ganglia together support a role for sensitized afferent nerve terminals in BO. Overall, genomic changes in efferent and afferent neurons of bladder induced by the overexpression of BDNF per se establish a mechanistic link between elevated BDNF levels in urine and dysfunctional voiding observed in animal models and in OAB patients.