FK506 Binding Protein 12 Is Expressed in Rat Penile Innervation and Upregulated After Cavernous Nerve Injury

FK506 binding protein 12 is expressed in rat penile innervation and upregulated after cavernous nerve injury

SF Sezen1, S Blackshaw2, JP Steiner3 and AL Burnett1*

1Department of Urology, The James Buchanan Brady Urological Institute, The Johns Hopkins Hospital, Baltimore, Maryland, USA; 2Department of Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA; and 3Guilford Pharmaceuticals, Baltimore, Maryland, USA

To evaluate whether FK506 and other immunophilin ligands may have potential therapeutic efﬁcacy for erectile function preservation after penile nerve injury, we demonstrated localizations of the immunophilin FK506 binding protein 12 (FKBP 12) in intact and injured rat penile nerves and correlated these ﬁndings with localizations of neuronal nitric oxide synthase (nNOS), which neuronally forms nitric oxide for mediation of penile erection, in response to systemically administered FK506. Adult male Sprague – Dawley rats were subjected to unilateral right cavernous nerve forceps crush injury and administered FK506 (1 mg=kg i.p.) or saline at the same time and daily up to 7 days. At 1, 3 and 7 days after injury, bilateral cavernous nerves and major pelvic ganglia were collected for nNOS immunohistochemistry, FKBP 12 immunohistochemistry, and FKBP 12 in situ hybridisation. Protein expressions of nNOS and FKBP 12 were observed in major pelvic ganglion, cavernous nerve and nerve terminals within the rat penis as well as mRNA expression of FKBP 12 observed in the rat major pelvic ganglion neuronal cell bodies to a minimal extent at baseline conditions. After cavernous nerve injury, nNOS immunoreactivity was observed to be slightly diminished in ipsilateral penile nerve structures at only one day following injury while both FKBP 12 protein and mRNA expressions were observed to be increased at each interval of study. FK506 treatment did not affect staining of intact or injured nerves. Our demonstration that FKBP 12 is localized to penile innervation in the rat and becomes upregulated following cavernous nerve crush injury, independent of FK506 treatment, suggests that this immunophilin mediates a neurotrophic mechanism. Whether FK506 affords neuroprotection that preserves penile erection through FKBP 12 upregulation is unclear. International Journal of Impotence Research (2002) 14, 506–512. doi:10.1038=sj.ijir.3900919

Keywords: immunophilin; neurotrophic; neuroprotective; nitric oxide

Introduction provided a foundation for subsequent characterizations of immunophilin ligands as neurotrophic and neuroprotective agents.6–13 FK506 and other immu- Immunophilins constitute a family of cellular nophilin ligands have been promoted as having proteins that act as receptors for immunosuppres- potential therapeutic efficacy for nerve recovery for sant drugs such as FK506 and cyclosporin A, whose such clinical presentations as neurodegenerative specific immunophilins are FK506 binding proteins diseases and nerve injury.14 – 17 1–3 (FKBPs) and cyclophilins, respectively. The We recently demonstrated that FK506 treatment of immunosuppressive effect resulting from FK506 rats protects penile innervation from degeneration treatment involves the binding of this drug to FKBP and preserves erectile function in a cavernous nerve 1–3 12 in immune cells. While FKBP 12 is expressed crush injury model.18 This report served as an initial in immune tissues, it is concentrated in much demonstration of the potential therapeutic role of higher amounts in neuronal structures such as the immunophilins for the neuroprotection of focally 4,5 brain. The finding that this immunophilin is so injured nerves important for penile erection that may significantly expressed in neuronal structures occur with radical prostatectomy.19 As preliminary work in this field, the report focused on a basic physiologic and anatomical description of the effects *Correspondence: AL Burnett, The Johns Hopkins Hospital, of FK506 treatment, monitoring intracavernosal 600 North Wolfe Street, Marburg 407, Baltimore, MD 21287-2411, USA. pressure responses to cavernous nerve electrical E-mail: [email protected] stimulation and nerve morphology by electron Received 25 May 2002; accepted 15 June 2002 microscopic analysis after cavernous nerve injury. FK506 binding protein 12 in rat penile nerve SF Sezen et al 507 The putative molecular mechanism for the FK506 with covalently attached FKBP 12 to affect affinity treatment effect in our rat cavernous nerve injury purification. Preabsorption of the antibody with model has not been reported earlier. Herein, we purified FKBP 12 protein abolished the specific demonstrate FKBP 12 localizations in intact and binding of the affinity purified antibody. Staining injured rat penile innervation and correlate these was performed with the Vectastain Elite ABC Kit findings with localizations of neuronal nitric oxide (Vector Laboratories, Burlingame, CA, USA) synthase (nNOS), which neuronally forms nitric peroxidase system, with diaminobenzidine (Dako oxide for mediation of penile erection. Corporation, Carpinteria, CA, USA) as the chromo- gen. The omission of primary antibodies provided negative controls. The grade of immunohistoche- Materials and Methods mical staining was assessed by two independent observers.

Experimental treatment In situ hybridization Studies were performed on tissues obtained following experimental treatment as described pre- Separate tissues were cryostat sectioned (20 mm viously.18 Brieﬂy, adult male Sprague – Dawley rats thick) for in situ hybridization as described (300 – 450 g, Charles River Laboratories Inc., previously.21,22 Digoxigenin labeled (Dig RNA Wilmington, MA, USA) were anesthetized with Labeling Mix, Boehringer Mannheim=Roche 1 pentobarbital sodium (Nembutal , Abbott Labora- Molecular Biochemicals, Indianapolis, IN, USA) tories, N. Chicago, IL, USA; 50 mg=kg i.p.) for RNA oligonucleotide probes (antisense, sense) were unilateral right cavernous nerve exposure and focal prepared from full-length (600 bp) rat FKBP 12 crush with Dumont no. 5 jeweler’s forceps three cDNA.22 Alkaline phosphatase-conjugated anti- times for 15 s 2 mm distal to the major pelvic digoxigenin antibody (Boehringer Mannheim= ganglion under aseptic conditions. FK506 (Prograf1, Roche Molecular Biochemicals, Indianapolis, IN, Fujisawa Healthcare Inc., Deerﬁeld, IL, USA; USA) and X-phosphate=nitroblue tetrazolium 1mg=kg i.p.) was administered at the same time as (Boehringer Mannheim=Roche Molecular Biochemi- the crush and on successive days, and penile cals, Indianapolis, IN, USA) were used for the erection and nerve morphology were evaluated 1, 3 detection of signal. Sense probe labeling were used or 7 days later. After completion of experimentation, as a negative control. Two independent observers animals were euthanized with pentobarbital sodium assessed the grade of staining. (250 mg=kg i.p.), and bilateral cavernous nerves, major pelvic ganglia and penis were collected for further investigation. All experiments were con- ducted in accordance with the Johns Hopkins Results University School of Medicine guidelines for care and use of animals. nNOS immunohistochemistry

Immunohistochemistry Protein expression of nNOS was observed in major pelvic ganglion, cavernous nerve, and nerve terminals within the rat penis, similar to earlier descrip- Upon removal, tissue specimens were immediately tions23 (Figure 1). In saline-treated animals, nNOS frozen in OCT Compound (Tissue-Tek, Sakura immunoreactivity was observed to be slightly more Finetek USA Inc., Torrance, CA, USA). Cryostat faint in the injured right cavernous nerve and major sections (6 mm thick) were cut, mounted on gelatin- pelvic ganglion compared with that of the contra- coated slides and ﬁxed with 4% paraformaldehyde. lateral structure at 1 day after right cavernous nerve Sections were processed with primary antibody injury, and otherwise no appreciable differences incubation as described previously,20 using a rabbit were observed at 3 and 7 days. In FK506-treated polyclonal antibody against human nNOS (amino animals, nNOS protein expression was unchanged acids 1419 – 1433) at a 1:1000 dilution (Diasorin1, in the major pelvic ganglion and in the cavernous Stillwater, MN, USA) and rabbit anti-human nerve both ipsilateral and contralateral to the polyclonal antibody against recombinant FKBP 12 cavernous nerve injury over the entire 7 day time at a 1:500 dilution. The FKBP 12 antibody was raised course. The retained nNOS expression in the major in New Zealand white rabbits against human pelvic ganglion in distributions ipsilateral to the recombinant FKBP 12, which was expressed in and cavernous nerve following FK506 treatment is puriﬁed from E. coli lysates. The immune polyclonal consistent with the neuroprotective effect demon- serum was then passed over a Sepharose column strated functionally by erection monitoring and

International Journal of Impotence Research FK506 binding protein 12 in rat penile nerve SF Sezen et al 508

Figure 1 nNOS immunohistochemistry of the major pelvic ganglion in saline and FK506-treated rats, one day after right cavernous nerve crush injury. In saline-treated animals, decreased nNOS immunoreactivity (-ir) is observed in cross sections of the ipsilateral ganglion (R-MPG) compared with the contralateral intact nerve side (L-MPG). In FK506-treated animals, similar nNOS-ir is observed in cross-sections of R-MPG and L-MPG. No primary antibody staining was used as a negative control (scale bar, 15 mm). Abbreviations: R-MPG, right major pelvic ganglion; L-MPG, left major pelvic ganglion.

histologically by electron microscopy.18 The absence interval of study. The observed increase in FKBP 12 of significantly depleted nNOS protein in the immunoreactivity following cavernous nerve injury ipsilaterally injured penile nerve distributions with parallels findings observed in models of facial and saline treatment may reflect the minor degree of sciatic nerve crush,24 suggesting that FKBP 12 is nerve injury and the short time course associated upregulated in response to cavernous nerve injury. with this experimental model. The results suggest that FK506 treatment does not significantly alter FKBP 12 protein upregulation following cavernous nerve injury, compared with the effect of saline treatment, at least in the short FKBP 12 immunohistochemistry term, although FK506 does apparently preserve functional erection during this time course.18 FKBP 12 protein expression was observed in rat major pelvic ganglion, cavernous nerve, and penile nerve terminals (Figure 2). In both saline-treated and FKBP 12 in situ hybridisation FK506-treated animals, FKBP 12 immunostaining in the major pelvic ganglion was observed to be more intense on the cavernous nerve injury side FKBP 12 mRNA expression was observed in the rat compared with the contralateral intact side at each major pelvic ganglion with localizations confined to

International Journal of Impotence Research FK506 binding protein 12 in rat penile nerve SF Sezen et al 509

Figure 2 FKBP 12 immunohistochemistry of major pelvic ganglion in saline and FK506-treated rats, one day after right cavernous nerve crush injury. In saline-treated animals, increased FKBP 12 immunoreactivity (-ir) is observed in cross-sections of the ipsilateral ganglion (R-MPG) compared with the contralateral ganglion (L-MPG). In FK506-treated animals, similar FKBP 12-ir is observed in cross-sections of R-MPG and L-MPG. No primary antibody staining was used as a negative control (scale bar, 15 mm). Abbreviations: R-MPG, right major pelvic ganglion; L-MPG, left major pelvic ganglion. neuronal cell bodies (Figure 3). Similar to FKBP 12 been shown to promote regeneration and functional immunohistochemical results, FKBP 12 mRNA recovery in in vivo animal models of peripheral expression was increased in the ipsilateral ganglion nerve injury such as crush injury of sciatic and associated with cavernous nerve injury compared facial nerves.8,11,13,24 We recently demonstrated that with the contralateral ganglion in both saline-treated FK506 exerts potent neuroprotective effects on rat and FK506-treated animals. These data further penile innervation and preserves erectile function in implicate augmented FKBP 12 expression in the a cavernous nerve crush injury model.18 In instances penile innervation in response to cavernous nerve of sciatic and facial crush nerve injury, FKBP 12 injury. localizations and upregulated expression have been confirmed and linked to nerve regeneration.24 To begin to characterize the mechanism of action of Discussion FK506 in penile innervation after cavernous nerve injury, we proceeded to perform localization studies for FKBP 12 in this innervation while correlating Recent descriptions of immunophilins in neuronal changes in expression of nNOS. We also examined structures have generated great interest in defining FKBP 12 expression in these injured structures with their likely roles in neurotrophic mechanisms.6 – 17 FK506 treatment. Our findings in this study, show- The prototype immunophilin ligand FK506 has ing that FKBP 12 is localized to penile innervation

International Journal of Impotence Research FK506 binding protein 12 in rat penile nerve SF Sezen et al 510

Figure 3 FKBP 12 in situ hybridisation of major pelvic ganglion in saline and FK506-treated rats, one day after right cavernous nerve crush injury. In saline-treated animals, increased FKBP 12 expression is observed in cross-sections of the ipsilateral ganglion (R-MPG) compared with the contralateral ganglion (L-MPG). Similarly in FK506-treated animals, increased FKBP 12 expression is observed in cross-sections of the ipsilateral ganglion (R-MPG) compared with the contralateral ganglion (L-MPG). Sense FKBP 12 cDNA probe was used as a negative control (scale bar, 15 mm). Abbreviations: R-MPG, right major pelvic ganglion; L-MPG, left major pelvic ganglion.

in the rat with apparently upregulated expression the role of immunophilins in penile nerve function following cavernous nerve crush injury, indepen- recovery. Findings from this study can also be dent of FK506 treatment, suggest that this immuno- extrapolated to the human condition such as trauma philin mediates a neurotrophic mechanism. involving penile innervation following radical pros- Whether the neuroprotective effects of FK506 in tatectomy.19 In rats, similar to humans, penile our model operate through FKBP 12 upregulation autonomic innervation derives from the major remains unclear. pelvic ganglion, termed pelvic plexus in humans, The in vivo rat model of penile erectile function is located bilaterally dorsolateral to the prostate.28 an ideal one to establish further a role for immuno- Cavernous nerve fibers arising from this ganglion philins in functional nerve recovery. This model innervate the erectile tissue of the penis, and its affords an experimentally advantageous neurophy- distributions contain nitric oxide synthase (NOS), siological system for purposes of manipulation and which constitutively catalyzes the generation of the quantitation. We have been able to advance earlier- gaseous molecule nitric oxide, identified to be the described neurophysiological and morphologic principal neuroactive mediator of penile erection.29 findings after cavernous nerve injury23,25 – 27 with Several previous studies have been carried out to the addition of a molecular study that characterizes investigate nerve injury and regenerative properties

International Journal of Impotence Research FK506 binding protein 12 in rat penile nerve SF Sezen et al 511 in the cavernous nerve, particularly in relation to its neurotrophic role, although the consistent level the production and release of nitric oxide. In rats, of FKBP 12 upregulation with FK506 treatment does unilateral cavernous nerve neurotomy causes not clarify whether or how it mediates FK506 depletion of NOS in penile nerves and concomitant neuroprotection. Studies are ongoing to investigate decrease in electrically stimulated intracavernosal additional FKBP receptor subtypes and receptor pressure responses, whereas after 6 months intraca- mechanisms for FK50634 and roles of non-immuno- vernosal pressure responses are significantly suppressant immunophilin ligands that may serve recovered in association with regeneration of NOS- to preserve penile erection and other nerve- containing nerves.25 Unilateral cavernous nerve mediated pelvic functions in the face of neurological freezing has also been shown to damage NOS- disease or injury. containing nerve fibers, with a delayed recovery after treatment.26 More recent investigations have suggested that growth hormone and certain growth factors may serve as neurotrophic agents in the Acknowledgements regenerative process following cavernous nerve 30 – 33 injury in the rat. Supported by National Institute of Health grant DK Our investigation promoting a neurotrophic role 02568 (National Institute of Diabetes and Digestive and for immunophilins significantly supports an alter- Kidney Diseases) and National Kidney Foundation- native neurotrophic system besides conventionally Maryland Research grant. characterized neurotrophins that may be developed therapeutically for the treatment of nerve injuries and neurological disease. A unique aspect of immunophilin ligands is that they act via specific References receptors unlike that of classical neurotrophic mechanisms which may exert diverse and perhaps deleterious effects. Molecular mechanisms for the 1 Schreiber SL. Chemistry and biology of the immunophilins and their immunosuppressive ligands. Science 1991; 251: neurotrophic and neuroprotective actions of immu- 283 – 287. nophilin ligands remain unclear. It is known that 2 Schreiber SL, Crabtree GR. Immunophilins, ligands, and the the neuroregenerative properties of immunophilin control of signal transduction. Harvey Lect 1995; 91:99– 114. ligands such as FK506 do not involve calcineurin, 3 Marks AR. Cellular functions of immunophilins. Physiol Rev 1996; 76: 631 – 649. which is essential for immunosuppression, and non- 4 Steiner JP et al. High brain densities of the immunophilin immunosuppressant FKBP 12 ligands that fail to FKBP colocalized with calcineurin. Nature 1992; 358: inhibit calcineurin are neurotrophic.10 Ongoing 584 – 587. characterizations of different FKBP receptor 5 Dawson TM et al. The immunophilins, FK506 binding subtypes and other molecular targets subserving protein and cyclophilin, are discretely localized in the brain: relationship to calcineurin. Neuroscience 1994; 62: the action of immunophilin treatments in the future 569 – 580. may shed additional insight into regulatory mechani- 6 Sharkey J, Butcher SP. 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An immunosuppressant, FK506, results that were likely to be much more demon- protects hippocampal neurons from forebrain ischemia in the strative with a more significant degree of cavernous mongolian gerbil. Neurosci Lett 1996; 204: 157 – 160. nerve injury and perhaps long-term assessment. 10 Steiner JP et al. Neurotrophic immunophilin ligands stimulate Similarly, the limited alterations in NOS immu- structural and functional recovery in neurodegenerative animal models. Proc Natl Acad Sci USA 1997; 94: 2019 – 2024. noreactivity in penile innervation distinguished 11 Lee M, Doolabh VB, Mackinnon SE, Jost S. FK506 promotes 25,26 from that of previous studies may readily be functional recovery in crushed rat sciatic nerve. Muscle Nerve explained by the acute time course used in this 2000; 23: 633 – 640. investigation. However, our results would indicate 12 Freeman EE, Grosskreutz CL. 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