*Division of Toxicological Sciences and Departments of *Neuroscience, §Pharmacology ...... grant from the International Life Sciences Institute, and the W. M..
Proc. Nati. Acad. Sci. USA Vol. 91, pp. 3191-3195, April 1994
Neurobiology
Immunosuppressant FK506 promotes neurite outgrowth in cultures of PC12 cells and sensory ganglia (cydophlifn/mmnoplin/nere growth fnctor/neurodgnermton/neurnl regeneration)
W. ERNEST LYONS*, EDWIN B. GEORGEt, TED M. DAWSONtt, JOSEPH P. STEINERt, AND SOLOMON H.
SNYDER§1II1
*Division of Toxicological Sciences and Departments of *Neuroscience, §Pharmacology and Molecular Sciences, Psychiatry and Behavioral Sciences, and tNeurology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205
Contributed by Solomon H. Snyder, January 3, 1994
examined the effects of FK506 on neurite extension in PC12 cells and sensory ganglia. We report dramatic neurotrophic actions of FK506.
The Immunosuppressant drug FK506 acts by ABSTRACT binding to receptor proteins, FK506-binding proteins (FKBPs), which in turn can bind to and regulate a Ca2+-dependent phosphatase, calcineurin, and a Ca2+ release channel, the ryanodine receptor. Based on our fins in regeneration models that levels of FKBPs during neural regeneration parallel those of growth-aociated protein GAP43, a calcineurin substrate that regulates neurite extension, we examid effects of FK506 in PC12 rat pheochromocytoma cells and in rat sensory ganglia. FK506 enhances neurite outgrowth in both systems by increaing sensitivity to nerve growth factor. Blockade of FK506 actions in sensory galka by rapamycin, an FK506 antagonist, establishes that these effects involve FKBPs. Rapamycin itself stimulates neurite outgrowth in PC12 cells. These drug effects are detected at sub omolar concentrations, suggesang therapeutic application in diseases involving neural degeneration.
The immunosuppressant drugs cyclosporin A and FK506 are thought to exert their therapeutic effects by binding to receptor proteins designated cyclophilins and FK506-binding proteins (FKBPs) respectively. When complexed to the immunosuppressant drugs, these binding proteins, designated immunophilins, bind to the Ca2+-activated phosphatase calcineurin to inhibit its activity and increase levels of phosphorylated calcineurin substrate proteins (1-9). Concentrations of the immunophilins are far higher in the brain and peripheral nervous system than in immune tissues, and FKBP is colocalized with calcineurin throughout the brain, suggesting an important functional relationship (4). We recently showed that cyclosporin A and FK506 block the neurotoxicity elicited by glutamate acting at N-methyl-Daspartate (NMDA) receptors in cerebral cortical cultures (10). The mechanism for the neuroprotective effects of these drugs appears to be inhibition of calcineurin with an augmentation of phosphorylated levels of nitric oxide synthase (NOS) (10). Since phosphorylation of NOS inhibits its catalytic activity (11), the immunosuppressants effectively reduce NO formation, preventing the neurotoxic effects of NMDA in these cultures (12, 13). GAP43 is a prominent protein in neuronal processes associated with neurite extension and is also a major calcineurin substrate (14). Regeneration of damaged facial and sciatic nerves is associated with a marked augmentation of GAP43 mRNA levels (15-18). mRNA for FKBP increases in a close temporal correlation with GAP43 in these instances, implying a functional link between FKBP and GAP43 (W.E.L., T.M.D., J.P.S., and S.H.S., unpublished work). As these findings suggest a role for FKBP in neurite outgrowth, we
METHODS PC12 Cultures and Measurement of Neurite Outgrowth. PC12 rat pheochromocytoma cells were maintained at 370C and 5% CO2 in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated horse serum and 5% heat-inactivated fetal bovine serum. For differentiation in the presence of nerve growth factor (NGF), cells were plated at 105 per 35-mm culture well coated with rat tail collagen at 5 pug/cm2 and allowed to attach before the medium was replaced with DMEM supplemented with 2% horse serum, 1% fetal bovine serum, and NGF and/or FK506 or rapamycin. For quantitation of neurite outgrowth, three to four random photographs were made per well, and neurons bearing processes longer than 5 pam were counted. Experimental conditions were unknown by the photographer and cell counter. Four separate experiments were performed in duplicate for each data point presented. Neurites were identified and counted from "400 cells per photograph. Thus, neurite-bearing cells from 1200-1600 cells were counted per data point. Dorsal Root Ganglion Cultures and Neurite Outgrowth. Embryos at day 16 were removed from pregnant SpragueDawley rats and the dorsal root ganglia were dissected. Whole ganglion explants were cultured in collagen-coated 35-mm dishes (Falcon) with N2 medium (DMEM/Ham's F12, 1:1, supplemented with progesterone, selenium, insulin, putrescine, glucose, penicillin, and streptomycin) at 370C in a 15% CO2 environment. Sensory ganglia were treated with various concentrations of NGF and/or FK506 or rapamycin or anti-NGF antibody. Ganglia were observed every 2-3 days under phase contrast with an Olympus (New Hyde Park, NY) IMT-2 inverted microscope, and axon lengths were measured. The axonal field of each ganglion was divided into four quadrants, and the length of the longest axons in each quadrant was measured with an eyepiece micrometer. The average of these measurements was taken as the axon length for the ganglion. (3HJFK506 Binding and Autoradiography. Levels of FKBPs in PC12 cells were obtained from Scatchard analysis of rH]FK506 binding curves. Cultures were scraped from the culture wells and homogenized in 10 volumes of 50 mM Tris'HCl, pH 7.4/1 mM EDTA with phenylmethylsulfonyl fluoride at 100 pg/ml. The homogenate was centrifuged at 40,000 x g for 20 min at 40C. Protein was determined by the Coomassie blue G250 binding assay using bovine serum albu-
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Abbreviations: FKBP, FK506-binding protein; NGF, factor. IlTo whom reprint requests should be addressed.
3191
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Proc. Nadl. Acad. Sci. USA 91 (1994)
Neurobiology: Lyons et al.
3192
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Time in
963 (DuPont/NEN) for measurement of radioactivity in a Beckman scintillation counter. Specific binding was determined by subtracting binding obtained in the presence of 1 pM unlabeled FK506 from total PHIFK5O6 bound. For PHJFK5O6 autoradiography, dorsal root ganglion cultures were grown on chamber slides coated with collagen at 5 pg/cm2. Cultures were fixed on the slide with ice-cold 4.0% freshly depolymerized paraformaldehyde in 0.1 M sodium phosphate (pH 7.4) for 1 hr and then washed twice with phosphate-buffered saline. Fixed cultures were labeled with [3HJFK506 by preincubation of the slides in 50 mM Hepes/ 0.2% bovine serum albumin/0.02% Tween 20 at pH 7.4, followed by incubation in the same assay buffer containing 1 nM [3H]FK506. Nonspecific binding was determined by adding 1 p.M unlabeled FK506. The slides were then rinsed four times for 5 min prior to drying and juxtaposed to 3H-sensitive film for 10 days.
100
culture, hr
FiG. 1. pH]FK506 binding in PC12 cells maintained in the presence or absence of NGF (50 ng/ml). n = 3 for each time point. Bars represent SEM.
RESULTS by PC12 Cells Conti FKBPs Whe Leves Are En NGF. We examined PC12 cells for the presence of FKBPs by monitoring the binding of VH]FK506 to cells under basal conditions and after treatment with NGF (Fig. 1). [3H]FK506 bound saturably to untreated PC12 cell homogenates. In typical experiments about 1000 cpm were bound; nonspecific binding in the presence of 1 pM FK506 was about 150 cpm. Fifty percent inhibition of PH]FKSO6 binding occurred with
min as a standard. Binding of 250 pM 3H]dihydro-FK506 (86.5 Ci/mmol, DuPont/NEN) was assessed for samples containing 5 pg of soluble protein in a final volume of 0.4 ml of assay buffer containing 50 mM TrisHCl (pH 7.4), 0.2% bovine serum albumin, and various concentrations of unlabeled FKS06. After 60 min at 25VC, 0.35 ml was layered over a 0.8-ml column of LH-20 Sephadex (Pharmacia LKB) equilibrated with assay buffer. The column was washed with 0.4 mi ofassay buffer, and the eluates were collected and mixed with formula C3 F 1.
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