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

Abstract

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 p75^NTR), 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.

Keywords: afferent pathways; bladder overactivity; bladder transfection; brain-derived neurotrophic factor; efferent pathways; overactive bladder.

Fig. 1.

Bladder wall transgene injection and validation of transfection experiments. A–C: control group was injected only with protamine complexed with liposomes without including the green fluorescent protein (GFP) plasmid. GFP protein was viewed at wavelength 515–550 nm, and the corresponding images of the DAPI counterstain were taken at wavelength 418–473 nm channel. D–I: dose-dependent increase in green fluorescence in bladder wall after transfection of GFP plasmid complexed with protamine and cationic liposomes validates the transfection procedure followed for BDNF and luciferase gene in subsequent experiments. Images were taken with Olympus fluorescence upright microscope model Bx51 (Olympus) and merged using software MagnaFire version 2.1 provided. Magnification is indicated by scale bar.

Fig. 2.

Bladder wall transfection of brain-derived neurotrophic factor (BDNF) transgene caused BDNF overexpression. A and B: BDNF plasmid (A) complexed with protamine sulfate and liposomes (total of 10 µg of plasmid in 80-µl volume) was injected in 20-μl volume/site and at 4 separate sites (anterior, posterior, and bilateral) between bladder neck and middle using a 30-gauge needle (B, image i). Hematoxylin and eosin staining of bladder from both groups does not show any structural abnormalities (B, images ii and iii). C–F: high levels of BDNF protein in bladder tissues (C) together with the lack of any change in BDNF levels of kidney tissues (D) and in serum (E) indicate that urine elevation can be traced to the exogenous overexpression in bladder (F). H: the lower bladder tissue levels of nerve growth factor (NGF) after BDNF transfection further confirm the critical role of BDNF in the observed physiological findings. I: quantitative real-time PCR of bladder tissue harvested 7 days after bladder wall transfection revealed significant upregulation of the genes critical for the enhanced BDNF signaling viz., Trkb, P75NTR, Plcg1, and plcg2 in the BDNF group. *P < 0.05, **P < 0. 01, and *** P < 0.001; unpaired Student’s t-test.

Fig. 3.

BDNF overexpression causes bladder overactivity (BO). Cystometry (CMG) performed under urethane anesthesia revealed that BDNF overexpression significantly reduced the intracontraction intervals (ICI; A and B) and compliance (C) but increased the amplitude of maximum voiding pressure (D); n = 5. *P < 0.05, **P < 0. 01, and ***P < 0.001; unpaired Student t-test). ■, Individual data points of the luciferase group; ▲, individual data points of the BDNF group.

Fig. 4.

BDNF overexpression causes genomic changes in cholinergic neurotransmission. Real-time PCR analysis showed that transcript of genes specific for cholinergic neurons such as choline acetyltransferase (ChAT), vesicular acetylcholine transporter (VAChT), Oct2, Adrα1a, Adrα1b, and Adrα1d was significantly upregulated in the BDNF group (*P < 0.05, **P < 0. 01, and ***P < 0. 001; unpaired Student’s t-test). Relative expression of Gja1 (Connexin-43) and M2 receptor was also increased in BDNF group without any change in the expression of Gjc1 (Connexin-45), and M3, receptor isoforms.

Fig. 5.

Immunohistochemical analysis of BDNF-induced genomic changes. Immunohistochemical analysis confirmed the BDNF-induced upregulation of ChAT, VAChT, M2 receptor (M2), and Connexin-43 (Cx43) genes. The higher expression of ChAT and BDNF was colocalized in rat detrusor smooth muscle. The double staining for VAChT is shown together with Cx43. BDNF selectively increased the expression of M2 and Cx43, whereas the expression of M3 receptor (M3), gap junction protein Connexin-45 (Cx45), and β₃-adrenergic receptor (β₃AR) remained unchanged. Alexa fluor 488 staining was viewed in the 515- to 550-nm channel, whereas Alexa flour 594 was viewed in the 604- to 644-nm channel, and the corresponding DAPI counterstain images were taken in the 418- to 473-nm channel. Magnification is indicated by scale bar. U, urothelium; SM, smooth muscle.

Fig. 6.

Functionality of enhanced cholinergic neurotransmission in bladder. A and B: representative tracings of electrical field stimulation (EFS; 1–64 Hz; 20 V, 5-ms pulses for 2-s duration) evoked contractions in urothelium intact bladder strips from luciferase- and BDNF-transfected groups. C: comparison of normalized amplitude at each stimulation frequency showed significantly increased responses at 16, 32, and 64 Hz in the BDNF group. D and E: representative tracings for the frequency-response curve in the presence of 1 µM neostigmine. F: comparison of peak contraction amplitude normalized to wet strip weight at respective stimulation frequency in the presence of neostigmine showed a significantly increased response in the BDNF group from 4 to 64 Hz. (*P < 0.05, **P < 0. 01, and ***P < 0.001; 2-way ANOVA, followed by Bonferroni’s post hoc test).

Fig. 7.

Atropine-sensitive EFS contractile response. A: the ACh release evoked by EFS is dominant at >10 Hz, and the consequent contractile response is sensitive to the muscarinic receptor antagonist atropine. B: the peak contractile response normalized to strip weight in the BDNF group was significantly elevated (2.81 ± 0.30 mN/mg) relative to the luciferase group (1.25 ± 0.11 mg, P < 0.001) at the frequency of 20 Hz in the absence of atropine. In the presence of atropine, a significantly larger reduction of the peak contractile response was noted in BDNF group, as was evident from the larger Δ2 compared with Δ1 measured in the luciferase group (2-way ANOVA followed by Bonferroni’s post hoc test). Larger atropine sensitive component of evoked contractile response at 20 Hz in the BDNF group further confirms the findings obtained in the presence of neostigmine and led us to conclude that exogenous BDNF overexpression in bladder enhances the cholinergic neurotransmission, which is the primary mode of neurotransmission in the efferent arm of micturition reflex.

Fig. 8.

BDNF overexpression causes genomic changes in the afferent neuronal pathways. BDNF-induced BO involves BDNF induced genomic changes in the afferent pathways, as shown by the real-time PCR data, with significant (P < 0.05) upregulation of transient receptor potential cation channel, subfamily V, member 1 (Trpv1; 2.54 ± 0.19-fold), and transient receptor potential cation channel, subfamily A, member 1 (Trpa1; 5.74 ± 0.25-fold) in the bladder tissue of the BDNF group relative to luciferase group (A). A and B: increased expression of pannexin channels (Panx1; 38.74 ± 9.26-fold) is consistent with the significantly increased bladder tissue levels of ATP in the BDNF group relative to luciferase group (B) and the molecular evidence of sensitized afferent nerve terminals (A). C–H: BDNF overexpression also significantly raised the BDNF and NGF expression in S1 dorsal root ganglion without evoking any change in the expression of neurotrophins in midcervial ganglia, which is not innervated by bladder afferent neurons to suggest a role for enhanced afferent neuronal activity in BDNF-induced BO and enhanced retrograde transport of BDNF and NGF. (*P < 0.05 and **P < 0.01; unpaired Student’s t-test).