Urine samples of patients with OAB are characterized by decreased levels of NGF and high activity of the metalloproteinase 9 (MMP-9), the main protease digesting NGF into peptides. The urine from rodents with diabetic voiding dysfunction originating from type 1 diabetes present the same characteristics. Chronic treatment (4 weeks) with the p75NTR antagonist THX-B improves bladder functions and restore normal NGF levels in urine. This project aims to examine the expression of NGF and MMP-9 by bladder cells in culture and unravel the pathways used by THX-B to control the expression and synthesis of these proteins.

Urothelial and smooth muscle cells were grown from rat bladders after collagenase type IV digestion. Proteins from cell extracts were analyzed by PCRs, immunoblotting, ELISA and enzymatic kits. Confocal immunohistochemistry was performed on cells grown on coverslips. Crispr-Cas9 was used to knockdown MMP-9.

NGF and MMP-9 mRNA were expressed in urothelial and smooth muscle cells. Both cell types were major sources of proNGF and NGF, while MMP-9 enzymatic activity was much higher in urothelial cells. Immunohistochemistry localized both proteins in the cytoplasm of cells. Crispr-Cas9 successfully abolished the expression of MMP-9, leading to a potent decrease in MMP-9 activity and concomitant increase in the secretion of NGF without affecting proNGF levels. On the other hand, incubation with THX-B (5 µg/mL) had no effect on SMCs. However, THX-B increased secretion of NGF by urothelial cells, increasing the ratio NGF/proNGF, and potently decreased synthesis and secretion of MMP-9. Interestingly, α2-macroglobulin, a protein that binds proNGF and leads to sustain activation of p75NTR receptor was also decreased by THX-B. When grown on filter separating an upper and lower chamber to mimic the in vivo conditions, urothelial cells produced more NGF toward the upper than lower chamber. In the presence of THX-B, NGF secretion was increased solely toward the basal medium. Finally, THX-B was not linked to the pathways usually associated to p75NTR, including Erk, Jnk, P38 and phosphodiesterase-4D.

Cells of the bladder (both urothelial and smooth muscle) are major sources of NGF and MMP-9. The latter is the major enzyme determining the survival of NGF in the cell media. THX-B protects NGF by decreasing the expression of MMP-9, improving the balance between pro-apototic and survival signaling pathways elicited by neurotrophins and their precursors.

Bladder cells express and secrete NGF, proNGF and MMP-9. MMP-9 activity is directly linked to the amount of NGF in the cell media. THX-B increases NGF secretion from urothelial cells, at least in part by decreasing MMP-9 activity.