Immunophilin Regulation ofNeurotransmitter Release

Joseph P. Steiner,*t Ted M. Dawson,* Majid Fotuhi,§ and Solomon H. Snyder*I1#Departments of *Neuroscience, tNeurology, I'Pharmacology andMolecular Sciences, and #Psychiatry and Behavioral Sciences, JohnsHopkins University School of Medicine, Baltimore, Maryland, U.S.A.tDepartment of Neurobiology, Guilford Pharmaceuticals Inc.,Baltimore, Maryland, U.S.A.§Department of Neuroscience, Harvard University School of Medicine,Boston, Massachusetts, U.S.A.

ABSTRACT

Background: The immunophilins are proteins that me-diate actions of immunosuppressant drugs such as FK-506 and cyclosporin A by binding to calcineurin, inhib-iting its phosphatase activity, and increasing thephosphorylation level of transcription factors requiredfor interleukin 2 formation. Though concentrations inthe brain greatly exceed levels in immune tissues, nofunction has been previously established for nervoussystem immunophilins. Nitric oxide (NO) has been impli-cated in neurotransmitter release. FK506 appears to inhibitNO production by maintaining NO synthase in a highlyphosphorylated and thereby inactivated state. Accordingly,we examined effects of FK506 and cydosporin A on neu-rotransmitter release in PC12 cells treated with nervegrowth factor (NGF) and in rat brain striatal synaptosomes.Materials and Methods: We monitored effects of immu-nophilin ligands on [3H]l-neurotransmitter release fromPC12 cells differentiated with NGF. Rat brain striatal syn-aptosomes were loaded with radiolabeled transmitters andtreated with FK506 or cyclosporin A prior to initiatingneurotransmitter release with N-methyl-D-aspartate(NMDA) or potassium depolarization. Striatal synapto-somes were also loaded with 32P-orthophosphate andtreated with FK506. 32P-labeled synaptic veside proteinswere isolated from these synaptosomes in an attempt to

relate specific FK506-dependent phosphorylation of vesideproteins with the effects of FK506 on neurotransmitterrelease. Identification of proteins targetted by FK506 wasmade by immunoblot analysis and immunoprecipitation.Results: Low nanomolar concentrations of the immu-nosuppressant drugs FK506 and cyclosporin A (CsA)inhibit transmitter release from PC-12 cells and fromNMDA-stimulated brain synaptosomes. By contrast, theimmunosuppressants augment depolarization-inducedtransmitter release from synaptosomes. Synapsin I, asynaptic vesicle phosphoprotein, displays enhancedphosphorylation in the presence of FK506.Conclusions: Inhibition of transmitter release in PC- 12cells and NMDA-treated synaptosomes by inununosup-pressants may reflect augmented phosphorylation of NOsynthase, reducing its catalytic activity. This fits with therequirement of NO for transmitter release in PCI2 cellsand NMDA-treated synaptosomes. Stimulation by im-munosuppressants of transmitter release in potassiumdepolarized synaptosomes may result from augmentedphosphorylation of synapsin I, whose phosphorylation isknown to facilitate transmitter release. Thus, immunophi-lins may modulate release of numerous neurotransmittersboth by influencing NO formation and the phosphorylationstate of synaptic veside-associated proteins.

INTRODUCTIONThe immunophilins cyclophilin and FK506-binding protein (FKBP) mediate the immuno-

Address correspondence and reprint requests to: SolomonH. Snyder, Departments of Neuroscience, Pharmacology,Molecular Sciences, Psychiatry, and Behavioral Sciences,725 N. Wolfe St, Johns Hopkins University School of Medi-cine, Baltimore, MD 21205, U.S.A.

Copyright 1996, Molecular Medicine, 1076-1551/96/$10.50/0Molecular Medicine, Volume 2, Number 3, May 1996 325-333

suppressant actions of drugs such as cyclosporinA (CsA) and FK506 (1). The immunophilins oc-cur in substantially higher concentrations in thebrain than in the immune tissues and are highlylocalized in discrete neuronal populations to-gether with the calcium/calmodulin-activatedphosphatase, calcineurin (2). Complexes of theimmunosuppressants with the immunophilins

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bind to and inhibit the catalytic activity of cal-cineurin, thus increasing the phosphorylation ofproteins that are calcineurin substrates (2,3).FK506 and CsA potently and selectively blockneurotoxicity elicited by stimulation of the N-methyl-D-aspartate (NMDA) subtype of gluta-mate receptors, which have been implicated inneuronal damage associated with strokes andneurodegenerative diseases (4). This neuropro-tective action appears related to enhanced phos-phorylation of nitric oxide synthase (NOS)which inhibits NOS catalytic activity (4), as NOSinhibitors block NMDA neurotoxicity (5).

Despite the high levels of brain immunophi-lins and their involvement in neurotoxicity, aphysiologic role has remained elusive. The linkbetween immunophilins and NOS suggested apossible participation in neurotransmitter re-lease. Nitric oxide (NO) regulates neurotransmit-ter release, as NOS inhibitors block transmitterrelease in brain synaptosomes and PCi2 cells(6,7). Neurotransmitter release is also regulatedby the phosphorylation state of synaptic vesicleproteins like synapsin I, whose enhanced phos-phorylation is associated with increased trans-mitter release (8-10). In the present study, weshow that very low concentrations of FK506 andCsA regulate release of several neurotransmittersin PC12 cells and brain synaptosomes, effectswhich may reflect influences on phosphorylationof NOS and synaptic vesicle proteins. These ac-tions implicate the immunophilins in transmitterrelease physiology.

MATERIALS AND METHODS[3HJ-Neurotransmitter Release Assays

PC12 CELLS. PC12 cells were grown in the pres-ence of 50 ng/ml nerve growth factor (NGF) for8-10 days, as described previously (7). At 16-18hr prior to release assay, the cells were placed inDulbecco's Modified Eagle's Medium (DMEM)plus NGF and 20 ,uM choline chloride at 106cpm/ml. The cells were then washed three timeswith oxygenated 37° C neurotransmitter release(NTR) buffer (124 mM NaCl, 5 mM KCl, 1.5 mMNaHPO4, 2 mM MgCl2, 2 mM CaCl2, 6 mM glu-cose, 25 mM HEPES, pH 7.25) and resuspendedin 1 ml NTR buffer. Nitro-L-arginine or FK506was added for 5 min at 370C followed by additionof 40 mM KCl to stimulate acetylcholine (ACh)release. After 2 min the release process wasstopped by removing the supernatants and cen-

trifuging for 5 min at 12,000 X g. The superna-tants were phosphorylated with choline kinase(2 milliunits/ml) to facilitate the separation of[3H]-ACh from [3H]-phosphocholine. As a con-trol for nonspecific release or leakiness of thecells, supernatants from washed cells incorpo-rated with [3H]-choline were removed just priorto addition of KCl. These samples were processedidentically as the assay samples. Values obtainedfrom these control cells were then subtractedfrom the assay samples to determine specificneurotransmitter release. ACh release assayswere performed in triplicate or quadruplicate inthree separate experiments and data presentedare mean values.

[3H]-dopamine release was assayed similarlyto [3H]-ACh release, except that 8- to 10-dayNGF-treated PC12 cells were treated with [3H] -dopamine 60 min prior to assay. Release of do-pamine was stimulated by addition of 40 mMKCl for 2 min at 370C. Cell supernatants werethen centrifuged for 5 min at 12,000 x g, andradioactivity was measured in the supernatants.

RAT BRAIN STRIATAL SYNAPTOSOMES. Striatal synap-tosomes were isolated in 0.32 M sucrose after1,000 X g centrifugation of striatal homogenatesprepared with a Teflon homogenizer. The super-natant was diluted 2-fold with NTR buffer andincubated at 37° C for 15 min. Crude synapto-somes were then loaded with [3H]-neurotrans-mitter at 1 ,uCi/ml for 30 min at 370C, washedwith NTR buffer and recentrifuged at 1200 X gfor 15 min. Synaptosomes were preincubatedwith FK506 or rapamycin for 5 min at 370C.After pretreatment, KCl or NaCl was added to 40mM final concentration to evoke neurotransmit-ter release or provide a control, respectively. Af-ter 2 min of release at 370C, synaptosomes werecentrifuged at 10,000 x g for 5 min, and released[3H] -neurotransmitter was recovered in the su-pernatant. Nonspecific release of transmitter wasestimated from [3H] -transmitter recovered in thesupernatant of synaptosomes centrifuged beforeaddition of KCl or NaCl. Assays were performedin triplicate in three separate experiments andmean values are reported.

Phosphorylation of synaptosomal proteinsSynaptosomes isolated from 0.32 M sucrose ho-mogenization were incubated for 90 min with32P-orthophosphate (0.1 mCi/ml) in phosphory-lation buffer (26 mM NaHCO3, 124 mM NaCl, 5mM KCl, 2 mM CaCd2, 2 mM MgCl2, 10 mM

J. P. Steiner et al.: Immunophilins and Neurotransmitters

glucose, pH 7.2). The synaptosomes werewashed three times in phosphorylation bufferand recovered by centrifugation at 10,000 X g for15 min. Synaptosomes were then preincubatedwith 100 nM FK506 or buffer for 5 min at 37° C.After pretreatment, KCl was added to 40 mM tostimulate transmitter release and phosphoryla-tion. After 2 min of phosphorylation, synapto-somes were recovered by centrifugation at10,000 X g for 5 min. Pellets were resuspendedin ice cold H20 + 50 mM NaF and 10 AM sodiumpyrophosphate (to prevent dephosphorylation)for 30 min at 40C, followed by a 10,000 X gcentrifugation to remove the lysed synapto-somes. The 10,000 X g supernatant was thencentrifuged at 200,000 X g for 2 hr through a 150mM sucrose cushion to recover 32P-labeled syn-aptic vesicles. 32P-labeled synaptic vesicle pro-teins were separated by SDS-polyacrylamide gelelectrophoresis using the buffers of Laemmli(11), the gels dried down and autoradiogramswere prepared.

For immunoblot analysis, proteins from rep-lica SDS-gels were electrophoretically transferredto nitrocellulose and probed with affinity-puri-fied anti-synapsin I Ig (graciously provided byDr. Pietro DeCamilli, Yale University School ofMedicine).

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RESULTSFK506 very potently inhibits both the spontane-ous as well as the potassium depolarization in-duced release of [3H] -dopamine and [3H]-AChfrom PC 12 cells that have been differentiated byNGF treatment (Fig. 1). Effects on depolariza-tion-induced dopamine release are evident withas little as 0.1 nM FK506, while 1 nM FK506diminishes spontaneous and potassium depolar-ization-induced release of both dopamine andACh. Maximum inhibition is evident at 10 nMFK506 with a reduction of basal release to 50-

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FIG. 1. FK506 inhibits release of acetylcholine(ACh) and dopamine from PC12 cellsPC12 cells were cultured and [3H]-neurotransmitterswere released as described in Materials and Meth-ods. Various concentrations of FK506 or FK506 + 1

mM rapamycin were added to the cells 5 min priorto addition of NaCl or KCl to initiate transmitter re-lease. Release of [3H]-ACh (upper panel) and dopa-mine (lower panel) was quantified as described inMaterials and Methods. The amount of transmitterreleased by addition of NaCl (basal release, diago-nally lined bars) in absence of drug treatment wasdesignated 100% of control. Neurotransmitter re-leased by addition of KCI is indicated by white bars.*Statistically significant differences between drugtreatment and controls (p < 0.05 using paired andunpaired Student's t tests).

60% of control levels and reduction of depolar-ization-induced release to 35-40% of control.Release of dopamine and ACh from NGF-treatedPC 12 cells is also inhibited by NOS inhibitors asdescribed previously (7), confirming a role forNO in the release process. The immunosuppres-

PC12 cells-Dopamine release

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328 Molecular Medicine, Volume 2, Number 3, May 1996

sant rapamycin blocks FK506 binding to its re-

ceptor protein FKBP-12 and reverses numerouseffects of FK506 (12). Rapamycin (1 pgM) re-verses influences of FK506 on both dopamineand acetylcholine release. CsA (1 ,uM) also in-hibits both spontaneous and depolarization-in-duced release of dopamine.

In synaptosomes prepared from the corpus

striatum of rat brain, FK506 blocks NMDA-in-duced release of [3H]-glutamate, resembling ef-fects in PC12 cells (Fig. 2). FK506 fails to block[3H]-glutamate release from synaptosomes elic-ited by potassium depolarization (data notshown). Nitro-L-arginine, like FK506, inhibitsNMDA-induced [3H]-glutamate release in syn-aptosomes but not depolarization elicited release.Recently, Hultsch et al. (13) noted that FK506inhibits serotonin release from the rat basophilicleukemia cell line RBL-2H3, a mast cell model.We confirmed these findings and also noted thatnitro-L-arginine inhibits this release process (datanot shown).

The inhibition of neurotransmitter release bynitro-L-arginine confirmed our earlier findingsindicating that NO regulates neurotransmitterrelease in PC12 cells and synaptosomes (7). In-hibition of neurotransmitter release is elicited ina stereospecific fashion by isomers of N-methylarginine and is reversed by L-arginine (7). Potas-sium depolarization induced release of neuro-transmitters in PC 12 cells does not appear until 8days following NGF treatment coincident withthe first appearance of NOS immunoreactivityand enzyme activity (7,14,15). FK506 enhancesthe phosphorylation of NOS which inhibits NOScatalytic activity (16), accounting for the abilityof FK506 to block NMDA-neurotoxicity in corti-cal cultures (4). We suggest that inhibition ofneurotransmitter release in PC 12 cells by FK506and CsA similarly derives from enhanced phos-phorylation of NOS with inhibition of NOformation.

In PC 12 cells, where the release of transmit-ters is due to nitric oxide production, FK506markedly inhibits potassium-induced ACh anddopamine release (Fig. 1). However, in striatalsynaptosomes, FK506 produces markedly differ-ent effects (Fig. 3). Both basal and potassium-in-duced release of [3H]-dopamine, [3H]-y-aminobu-tyrate (GABA), [3H] -glutamate, [3H]-serotonin,and [3H] -norepinephrine are enhanced by thedrug, with clear-cut effects at 1 nM concentration,some enhancement at 0.1 nM, and maximal effectsat 10-100 nM. FK506 promotes the calcium-de-pendent release of neurotransmitters from striatal

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synaptosomes, since removal of extracellular cal-cium abolishes both depolarization dependenttransmitter release and the effects of FK506 (data

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FIG. 3. FK506 enhances the basal and depolarization-dependent release of neurotransmitters fromstriatal synaptosomesStriatal synaptosomes were labeled with [3H]-dopamine, [3H]-norepinephrine, [3H]-serotonin, [3H]-GABA, or[3H]-glutamate all from Dupont-NEN (Boston, MA) at 1 mCi/ml for 30 min at 37° C, washed with NTR buffer andcentifuged at 1,200 x g for 15 min. Synaptosomes were preincubated with the indicated concentrations of FK506or FK506 + 1 mM rapamycin for 5 min at 37° C. After pretreatment, KC1 or NaCl was added to 40 mM final con-centration to evoke neurotransmitter release. After 2 min release at 370C, synaptosomes were centrifuged at10,000 x g for 5 min and released [3H]-neurotransmitter was recovered in the supernatant. The amount of trans-mitter released by addition of NaCl (basal release, black bars) in absence of drug treatment was designated 100%of control. Neurotransmitter released by addition of KCI is indicated by diagonal lined bars. *Statistically significantdifferences between drug treatment and basal and K+ controls as indicated (p < 0.05 using paired and unpairedStudent's t tests).

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330 Molecular Medicine, Volume 2, Number 3, May 1996

not shown). The enhancement of norepinephrinerelease by FK506 is reversed by 100 nM rapamy-cin, demonstrating the specificity of the FK506-FKBP interaction and implicating the immunophi-lin FKBP as a mediator of the effects of FK506.[3H]-norepinephrine release from hippocampaland cortical synaptosomes is also enhanced byFK506 (data not shown).

How might one account for the finding thatFK506 fails to inhibit potassium depolarization-evoked release of neurotransmitter from synap-tosomes whereas it blocks NMDA-induced re-lease? NOS neurons possess NMDA receptors(17) and normally respond to NMDA with en-hanced formation of NO, which would diffuse toadjacent nerve terminals to elicit transmitter re-lease. While NMDA acts only on a subset of thesynaptosomal population, potassium depolariza-tion would influence all synaptosomes includingthose that lack NMDA receptors and NOS, so thatany influence of FK506 on NO-regulated neuro-transmission would be obscured.

In our experiments, FK506 stimulates bothspontaneous and potassium depolarization-in-duced transmitter release in synaptosomes. Thiseffect might involve synaptic vesicle proteinssuch as synapsin I, synaptobrevin, and synapto-tagmin. Phosphorylation of synapsin I correlateswith enhanced neurotransmitter release (8,9). Ifphosphorylated proteins such as these are cal-cineurin substrates, then FK506 might enhancetheir phosphorylation, thereby facilitating trans-mitter release. In the crude synaptic vesicle frac-tion isolated from striatal synaptosomes, FK506augments the phosphorylation state of severalproteins including those with apparent SDS-polyacrylamide gel molecular weights of 50 kD,58-60 kD, a doublet of about 70-75 kD, 120 kD,and 350 kD (Fig. 4). Immunoblot analysis indi-cates that the 70- to 75-kD doublet in synapticvesicles is synapsin I, whose increased phosphor-ylation state correlates well with increased gluta-mate release. FK506 treatment of synaptosomesleads to a 30-40% increase in 32P-phosphateincorporation into vesicle-bound synapsin I. Im-munoprecipatation of 32P-phosphorylated syn-apsin I from striatal synaptosomes demonstratesthat synapsin I contains 40-50% more phos-phate following FK506 treatment (Fig. 4). In-creased phosphorylation of synapsin I and theseother synaptic vesicle proteins in the presence ofFK506 indicates that they are calcineurin sub-strates.

DISCUSSIONThe immunosuppressant drugs FK506 and cyclo-sporin A potently inhibit the release of neuro-transmitters from PC 12 cells and NMDA-evokedrelease from rat brain synaptosomes. In the caseof NGF-treated PC 12 cells, where release oftransmitter is dependent upon NO production,FK506 would inhibit NO production by inacti-vating NOS and reduce release of ACh and do-pamine. The NO-dependent NMDA-stimulatedrelease of glutamate from striatal synaptosomesis likewise blocked by FK506, presumably due toinhibition of NOS catalytic activity. Although themechanism of the effects of FK506 on the releaseprocess in these cells is unknown, our data areconsistent with the notion that the targets ofFK506 and CsA are drug-immunophilin com-plexes binding to and inhibiting the proteinphosphatase calcineurin.

NO apparently plays an inconsequential rolein potassium depolarization-evoked release ofneurotransmitters from rat brain synaptosomes.FK506 and CsA potentiate the release of thebiogenic amines and amino acid neurotransmit-ters from rat brain synaptosomes. The FK506-elicited transmitter release is blocked by rapamy-cin, indicating that FKBP participates in therelease process. The calcineurin substrate synap-sin I (18), whose phosphorylation is associatedwith transmitter release, is more highly phos-phorylated in response to FK506 treatment, sug-gesting that phospho-synapsin I mediates FK506enhancement of synaptosomal transmitter re-lease. Several other unidentified phosphopro-teins display elevated 32p incorporation in re-sponse to FK506 and may also participate in theFK506 effects.

Blockade of calcineurin may also interferewith vesicle recycling. In response to Ca2+ entrycalcineurin dephosphorylates several prominentnerve terminal proteins. Dynamin I, a phospho-protein with intrinsic guanosine triphosphatase(GTPase activity), is required for endocytosis andis a substrate for calcineurin (19,20). In nerveterminals, calcineurin may serve as a Ca2+-sen-sitive switch for depolarization-evoked synapticvesicle recycling (19,20). In this way, calcineurincould reset the nerve terminal for the next roundof depolarization-induced neurotransmitterrelease.

FK506 and CsA enhancement of neurotrans-mitter may have diverse physiologic conse-quences. Long-term potentiation (LTP) and long-term depression (LTD), which participate in

J. P. Steiner et al.: Immunophilins and Neurotransmitters

AUTORADIOGRAPHY

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FIG. 4. Phosphorylation of synaptosomal proteins(a) Striatal synaptosomal proteins were preincubated with 32P-orthophosphate and phosphorylation monitored inthe presence of 100 nM FK506. Synaptosomes were lysed, and the crude synaptic vesicle fraction was recoveredfollowing centrifugation as described in Materials and Methods. The 32P-labelled synaptic vesicle proteins from un-treated and 100 nM FK506-treated synaptosomes were resolved by gel electrophoresis, the gels were dried, and anautoradiogram was prepared. Immunoblot analysis of these samples probed with anti-synapsin I Ig demonstratedequivalent amounts of synapsin in each fraction. Molecular weight markers in kilodaltons are indicated. (b) 32p_labelled synapsin I was immunoprecipitated from phosphorylated striatal synaptosomes treated with FK506 orbuffer containing 0.01% ethanol (the vehicle for FK506) as described in Materials and Methods. Duplicate samplesof anti-synapsin I immunoprecipitates from control and FK506-treated synaptosomes were resolved by gel electro-phoresis, gels dried down, and an autoradiogram was prepared. Synapsin I is indicated by the arrow at 73-75 kDa.Molecular weight markers are indicated in kilodaltons.

learning and information storage, are regulatedby calcium-dependent phosphorylation/dephos-phorylation (21). Calcineurin has been impli-cated in the generation of LTD in hippocampalslices, as FK506 inhibits this process (22). Gen-eration of LTD requires activation of postsynapticNMDA receptors (23), which also increases glu-tamate release. Thus, inhibition of LTD by FK506may reflect altered release of neurotransmittersassociated with increased phosphorylation of cal-cineurin substrate phosphoproteins, includingNOS.

Transplant patients receiving FK506 or CsAdevelop a variety of side effects, such as hyper-

tension, which is thought to be due to increasedsympathetic tone (24). Enhancement of norepi-nephrine release by FK506 and CsA could ac-count for increased sympathetic tone and hyper-tension.

ACKNOWLEDGMENTSThis work was supported by USPHS Grant DA-00266, Research Scientist Award DA-00074(SHS), and postdoctoral fellowship MH10101(JPS). TMD was supported by USPHS CIDA NS04578 and the Beeson Scholar Program in Aging

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Research. We gratefully acknowledge Dr. Sam-uel Danishefsky for the gift of FK506, Wyeth-Ayerst for the gift of rapamycin, and Sandoz forcyclosporin A used in these studies. The experttechnical assistance of Roxanne Barrow is grate-fully acknowledged. We also thank Nancy Brucefor preparing the manuscript. Some of the au-thors own stock in and are entitled to royalties(JPS, TMD, SHS) from Guilford Pharmaceuticals,Inc., which is developing technology related tothe research described in this paper.

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Contributed by S. H. Snyder on January 29, 1996.

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