Proc. Nati. Acad. Sci. USAVol. 91, pp. 3191-3195, April 1994Neurobiology

Immunosuppressant FK506 promotes neurite outgrowth in culturesof 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, andtNeurology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205

Contributed by Solomon H. Snyder, January 3, 1994

ABSTRACT The Immunosuppressant drug FK506 acts bybinding to receptor proteins, FK506-binding proteins(FKBPs), which in turn can bind to and regulate a Ca2+-de-pendent phosphatase, calcineurin, and a Ca2+ release channel,the ryanodine receptor. Based on ourfins in regenerationmodels that levels of FKBPs during neural regeneration par-allel those of growth-aociated protein GAP43, a calcineurinsubstrate that regulates neurite extension, we examid effectsof FK506 in PC12 rat pheochromocytoma cells and in ratsensory ganglia. FK506 enhances neurite outgrowth in bothsystems by increaing sensitivity to nerve growth factor. Block-ade of FK506 actions in sensory galka by rapamycin, anFK506 antagonist, establishes that these effects involve FKBPs.Rapamycin itself stimulates neurite outgrowth in PC12 cells.These drug effects are detected at sub omolar concentra-tions, suggesang therapeutic application in diseases involvingneural degeneration.

The immunosuppressant drugs cyclosporin A and FK506 arethought to exert their therapeutic effects by binding toreceptor proteins designated cyclophilins and FK506-bindingproteins (FKBPs) respectively. When complexed to theimmunosuppressant drugs, these binding proteins, desig-nated immunophilins, bind to the Ca2+-activated phospha-tase calcineurin to inhibit its activity and increase levels ofphosphorylated calcineurin substrate proteins (1-9). Concen-trations of the immunophilins are far higher in the brain andperipheral nervous system than in immune tissues, andFKBP is colocalized with calcineurin throughout the brain,suggesting an important functional relationship (4). We re-cently showed that cyclosporin A and FK506 block theneurotoxicity elicited by glutamate acting at N-methyl-D-aspartate (NMDA) receptors in cerebral cortical cultures(10). The mechanism for the neuroprotective effects of thesedrugs appears to be inhibition of calcineurin with an aug-mentation of phosphorylated levels of nitric oxide synthase(NOS) (10). Since phosphorylation of NOS inhibits its cata-lytic activity (11), the immunosuppressants effectively re-duce NO formation, preventing the neurotoxic effects ofNMDA in these cultures (12, 13).GAP43 is a prominent protein in neuronal processes asso-

ciated with neurite extension and is also a major calcineurinsubstrate (14). Regeneration of damaged facial and sciaticnerves is associated with a marked augmentation of GAP43mRNA levels (15-18). mRNA for FKBP increases in a closetemporal correlation with GAP43 in these instances, implyinga functional link between FKBP and GAP43 (W.E.L.,T.M.D., J.P.S., and S.H.S., unpublished work). As thesefindings suggest a role for FKBP in neurite outgrowth, we

examined the effects of FK506 on neurite extension in PC12cells and sensory ganglia. We report dramatic neurotrophicactions of FK506.

METHODS

PC12 Cultures and Measurement of Neurite Outgrowth.PC12 rat pheochromocytoma cells were maintained at 370Cand 5% CO2 in Dulbecco's modified Eagle's medium(DMEM) supplemented with 10% heat-inactivated horseserum and 5% heat-inactivated fetal bovine serum. Fordifferentiation in the presence of nerve growth factor (NGF),cells were plated at 105 per 35-mm culture well coated withrat tail collagen at 5 pug/cm2 and allowed to attach before themedium was replaced with DMEM supplemented with 2%horse serum, 1% fetal bovine serum, andNGF and/or FK506or rapamycin. For quantitation of neurite outgrowth, three tofour random photographs were made per well, and neuronsbearing processes longer than 5 pam were counted. Experi-mental conditions were unknown by the photographer andcell counter. Four separate experiments were performed induplicate for each data point presented. Neurites were iden-tified and counted from "400 cells per photograph. Thus,neurite-bearing cells from 1200-1600 cells were counted perdata point.

Dorsal Root Ganglion Cultures and Neurite Outgrowth.Embryos at day 16 were removed from pregnant Sprague-Dawley rats and the dorsal root ganglia were dissected.Whole ganglion explants were cultured in collagen-coated35-mm dishes (Falcon) with N2 medium (DMEM/Ham'sF12, 1:1, supplemented with progesterone, selenium, insulin,putrescine, glucose, penicillin, and streptomycin) at 370C ina 15% CO2 environment. Sensory ganglia were treated withvarious concentrations of NGF and/or FK506 or rapamycinor anti-NGF antibody. Ganglia were observed every 2-3 daysunder phase contrast with an Olympus (New Hyde Park, NY)IMT-2 inverted microscope, and axon lengths were mea-sured. The axonal field ofeach ganglion was divided into fourquadrants, and the length of the longest axons in eachquadrant was measured with an eyepiece micrometer. Theaverage of these measurements was taken as the axon lengthfor the ganglion.

(3HJFK506 Binding and Autoradiography. Levels ofFKBPsin PC12 cells were obtained from Scatchard analysis ofrH]FK506 binding curves. Cultures were scraped from theculture wells and homogenized in 10 volumes of 50 mMTris'HCl, pH 7.4/1 mM EDTA with phenylmethylsulfonylfluoride at 100 pg/ml. The homogenate was centrifuged at40,000 x g for 20 min at 40C. Protein was determined by theCoomassie blue G250 binding assay using bovine serum albu-

Abbreviations: FKBP, FK506-binding protein; NGF, nerve growthfactor.IlTo whom reprint requests should be addressed.

3191

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

3192 Neurobiology: Lyons et al.

20 40 60 80 100Time in culture, hr

FiG. 1. pH]FK506 binding in PC12 cells maintained in thepresence or absence ofNGF (50 ng/ml). n = 3 for each time point.Bars represent SEM.

min as a standard. Binding of250pM 3H]dihydro-FK506 (86.5Ci/mmol, DuPont/NEN) was assessed for samples containing5 pg of soluble protein in a final volume of 0.4 ml of assaybuffer containing 50 mM TrisHCl (pH 7.4), 0.2% bovineserum albumin, and various concentrations of unlabeledFKS06. After 60 min at 25VC, 0.35 mlwas layered over a 0.8-mlcolumn of LH-20 Sephadex (Pharmacia LKB) equilibratedwith assay buffer. The columnwas washed with 0.4 miofassaybuffer, and the eluates were collected and mixed with formula

Proc. Nadl. Acad. Sci. USA 91 (1994)

963 (DuPont/NEN) for measurement of radioactivity in aBeckman scintillation counter. Specific binding was deter-mined by subtracting binding obtained in the presence of1 pMunlabeled FK506 from total PHIFK5O6 bound.For PHJFK5O6 autoradiography, dorsal root ganglion cul-

tures were grown on chamber slides coated with collagen at5 pg/cm2. Cultures were fixed on the slide with ice-cold 4.0%freshly depolymerized paraformaldehyde in 0.1 M sodiumphosphate (pH 7.4) for 1 hr and then washed twice withphosphate-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 1nM [3H]FK506. Nonspecific binding was determined byadding 1 p.M unlabeled FK506. The slides were then rinsedfour times for 5 min prior to drying and juxtaposed to3H-sensitive film for 10 days.

RESULTSPC12 Cells Conti FKBPs Whe Leves Are En by

NGF. We examined PC12 cells for the presence ofFKBPs bymonitoring the binding of VH]FK506 to cells under basalconditions and after treatment with NGF (Fig. 1). [3H]FK506bound saturably to untreated PC12 cell homogenates. Intypical experiments about 1000cpm were bound; nonspecificbinding in the presence of 1 pM FK506 was about 150 cpm.Fifty percent inhibition of PH]FKSO6 binding occurred with

IIIIC3F 1. n o/'I \61 5': -Ic. l-,"

'.. .......!..".""...ll,.s-...Iftf.. ... .-.....' `.i:.,. ..--..:.: ... .-;T- M 'n's

1: ,::..,: ;. .,v.. .:":."..:..Ii.-IV.... -1. .4'1.: :..:,.: -n2 .... .: ...:.. .-1 :1 C .:!:-:-s1zf-- n'.

.,W ,-..;X .:.* :V:. ..: :. :-:'..... ,.,.-: --- .:

':I.;,,,.. ...:. -!:. V ::,.: ,....:,,. :L.. '. :" i.: i. :.. ,.;:" :. .....N :1::::. .'A'J'. .7

..::, .i::,,i-:-, .: N. .:.. :.. " .. 1. ,f;". :,: '. ,- -.f,::' .. ..., :..' ...:. .: .::.:;. i. R::-_- -1:, '.,:: .:::!!::.:.' `11.1.,,... ,..-.-,:v: '!;.:' ,4 --:.Y.':.- :, .. .. :..'r,1. ..,';: .., :....:. .,. .- ;.,:!S r.f.:: -'f :':'. Y ..- ..f:;,:. = .. .: !,. .:::f. :.7...z -- ::,i::. ... :.!--:.:- ..'.:-,. :, i. ." , ;r .p. :;:.. :,:!-. j... .:

.f.;!:.;.:: .:: -11.111-11....a.!;:..! ;.., f: -!::.'.:.:. .... -.! %::::suspect? .!:jj.;..:: .." .f;:,::i :: ?. ,. ...,.:..=nW :.. .:: -f'.s 'l."'. ,:.::. ...,.I, ;,... ..:,:s:.

.... ,It ..:..- -:'M,:..- f,..- :':,!..-.!;..,.Z.ii;..M i,;.; .-.-- .iE.!. .1. I(.f :11 :-.. ;.i,.:. !:.. ., :_, ::' :;:!:: .:.-:M .1: :.;:f:.:: !:M. '.

.:; !.:." :.s::f,, .. fl; .f_ ::....-.-....... .:t.;.. _. .s. ... ..; :. ,:::::-.::.::..: tz.". ..:::.,=.:sW :...; 1'. ...!:- .z,-'.;_. :::M f% ::. -:,z,-: :--:-::...;t-.::- -:.:.: .: ,..:1 ;:f .: :M :.: ':f-;!_.

:- .:..:.:-...:. f:. :" .;Uzs:,. .: :..: -....1::1:,.;-::.-U ...-. :k,

..Ifn .: .:. .. :. 1. f, 'I. :: :, ...f. .f,. :-. -- ..:C , r ,:, ,- ,.::: :,,-,; ..... .1- ., .. 11 j. ,if !.:: f.11: '.' .. .: ...f. ,:..'..4 ;... !:.:, 1. .--..:.: ........ ..: ': :.:.:-- :,: ::,.:: :::: !.;: ...: .: .: 'i..-1'1.s;.i.,;: 4::4 ..... .1. "If, :.:. -t..' ..,;. ?;.,i: !.. ,. 11.. :,: n .i t.: q .;; :: Z, .:,.'.. 'f 1: .;. .:: .:.-..',!:' ... .: -..;-. ": ..:1 1W

:: ... .... !:. .:: ,:. :... :. - :;---: .f.: :.: .:f::::-- i-.i-;, :: '. .'. ::-- ,.:. .'.'i:` :f ': ." -. :: .,. -: --- I...:. :i.:,-:!. ::,fl,. ,t:: .: i. NN ". !'::,.:. If .. :, ., ::f:.:- "i .. :-- .: M. , .::.!:, t.--:..

.., .... .: .... sfpz ,, .:- .:.,... :.V. ...4-,.: --.. *K .. ..: .! .: ?..:

..:..:2..-rjfj:.,:..: .:s" ..,.:. :- -.,:::- :.7:' !-...

.:. '.', ., , .',:, .1:..... :.-. :..f:..'-: .: .'.: -, .: '.'.:.'.:,t n ... ,; t,.,: :s7 :'!...f .1 .f. .'. 1.: ,. .1, f. _-:il;: .... :. ;.::.,.,M1::, 11 " "

:...;i ", .. :.. '_..E.. :.; .: '. 1.11i::., .... ... .:. :. .... .:.. ...., .% :1,114:1... .7, -.,z, -:. .'- , %,l .. -:f .:. .P- .... :.1.' .: ::::t lf:..; ._, .'i.. S.- 1, :: I.. .. ': :; ..::. ::... .i.,4:s in `tts '.:-. :: ,;, f, .::: st:z- ff .:...:,i:;:. .: .. , .: 9: _.. .; -, .:-., ,z. :.. .....:-.':-, .: ::!.: :.. t::ff'..: ::_.. :,. .!:.. :i ,f :".: :.: .:: :;: " , :... .... .;: .I:- .-: .... :.11. .11::. -, -, .: ..: .Wi. 1:1 :;,..: ,:::::.. -A;, .f ::

.i :.:.:, ..:. :- --.it;l ::,.--:,zY.,::M , !;., 1.1:- ==;:f.; .z.: .,, ., .: ._ f.::s:..!;-- --. .- :: .. ':.'...;:: :: '!:. ..... :....... ...!.,. :,:.:.f;lI...... .:......... ::.:,;:.

:. :.:.:- , .. .:. .-.:' :- ;': mmg :f 'R ",.', i, ,.:;I ;f .. -.1-1.,f.....I.. ..::!... .:.. M ss::.::-'-: 1:.....",,,'R,.:: ::.;::. :' ,.: :: ,.: :: ,..:s.....:,f...-. :. :. :. 's.ff'i ," 'f'..'.;....

-- ,m -;.%, .f..:. --f.. -..:..4... -.:.:. .:,::::I.,..g. ::f:tj f;-:--. '-,:; .:fi..;_-.; -'..!X.,4f::. .::.,;,. i.lk -'i. -.1., .T.;.5L_ ." ..f:! .:f. 1N ..4:- ,-:11.. ;-.:.f;,.!!:: .4. zr=

-'. :.::;.;; .;;;.;;v--,=:.;- 1,4fsts::-,;;,:.:;-; zul..;;Mz;::f .:.:; - 4:=;, '4= .:;11:f. .1 R;. ::. ...... " 1, .:. 1.

.i:- ::: .i:. :1. .:.WW 11 ':M if! ;... ".1. :?.ni .= :41, -1I.. .... ;:: ::: .;; .2.,'_ :;.Iz

:...:;-:M;::,- :: -, ". ..:=:.; .: .!:: ::.:. ,- :; ::. .:1,::,,M ;I.f:".,f..s:.z 1,:. ;:::- f.t;;;.::.-:- -:. ..:!! .i:::-:-... %; .. ..2; s: :, .4:::: ::!ff.: V, !:-t., :.,:Us 11s ..: ,j;%::`f .:;:zs, .::tl .::tl:-:: ... .. .; :;:.:,:!:.1;::: .. :;.; ;,.:, :'...... ;:V;.I,,X,.`1.. ... :4 '? i'.'..-. -:--;:'-, "..;: -%.-` .: .:; ".1;,-:1;, ;;:-.,;; .-:_:.:, -.:. .: .. ...: ... :V.!::f:--,,f, .f .:.,. .,:. .,. ::. :f..:: :-' :s..f,,,:,:;'.s;.;::;1f.-. .. f. ,..W." W;it:""i::;`1` ': " ;.

,.:"% :.. .1 :,.Analytical f" ..: 1..f .. .. ..

, :f :I:- '.'.: !::-" .,: ,,, !:. .:i ,:; :;;::1 .. ..%.".: -:1.--._1117,:!... .:, .:. .:.,. :'! .1.1 :- ,: :;:

.-:,_fi: .;I-:. .: 'I- ., ...:: .1:f-. .:.. `r ->i. ... ..... -- :: .!:::,.,. ., ... .:.i.'; :. ;--,. ...... !- :,..,:: 1. :-;...,.:: '., ..: '-."

.":.:..::. .11'. .. f; f. .'. .,a-- .. .'.: :.

I.: '. f:..:. :, .:,i:::, .. ..4.;1 :...... - -:: :.:. ..-..I. .1 -. I.. ..

!,:, .?. " ,-:.!%,.- .7... ..

.. ., ;:--. :: ,:'::. ... .,.,.::,. :. .I_'. -. -. ." :, .,:.: ,, .!..:. 17. " .., .. .:,

:.

.-.:.'- .: ..: .. .1; ::;:. .:.0 ...:, .- 0."Ill. .. .. :...-V 't, --7-1:-:..- ..:.--.:. .I

;, -:`.;,:!:fM., :..:.. i.- Z-4t."j,-4 -... .:-.... ,!,..fl .:: ;- - i: ._ .:f :.

... .: .. - ... ... ,,,,.;,:f.; ..' , :.4::::;: -.114 P ....., ,.. ,.":.. 1.'- :: -,

..i.".,... :-'! .. ;. ..:. LTV :::---- .: :: ::::: .: --:. isp..-..:-.t:-::..:. M. .:11W.: :. !.:- :..f.::1 .: ,::;, ;-;. :.. 4:. :. ,::i.-;.:.:- ::. ,..., 1., M ;...'r,:.. .... U:: .. -,.- - ,;! .':, R"AIN: -;i .1., :;.: !.:.:; M ': ..;U.: .:f,. :4,:, .:: :;: R. .,i..: .-..:;..,.: ..... .. -4s;U,::!f:. .: ..; 4' 0.. :::: .:: f:: '. .:.: ;: :;. ..: -F

.::.4,.:!:.. ... ...& -:: f.:, .1. ,:,.:::: .:.: ., ... -i... t'.W, w.,.:.!:: .:..!:.f::.: ...... .MOIR 15ga R1,." 'is1. ::.. ;4-- .;, .iI

.,"." -:, :,.;:;.;Beaze ,.. .;;:.':f. ,..: ..:- ;, si;.:-:tfW ;--;.-,;.- -. .ou, '! .,. ,..'i, !..;.!!;:!.,:: '. -:.i:..IAN ,..i.i.: i,:, :SM M :1 .,.. t:;:f, ..s -:.'-;-,.-:.--:.:....:!;%. :. ,f'.;;.,: -:1" :-::::.:..!:f'-.: .1048.' 1.z-:1 .: .:.;.:z;-ff'.- .= ::.- f.!; :. '.'..s:_ ;.M 1'. ,.: .: .. ..-f ;.. ::: :'I :f" " :, ,.... :. ;f:: ..:,slrw*!,. :', :.. -Rr" ,. .7ffmw I..",.11 .v.'1:7 ..I,, .;,.t 1..?.,;:.-..:. f: .,. ..::.:t!;.::- .:,.,, :... .!:;;. .:fs,..f,:ji:..f, u: ': .. .j. .: ..?Z; ..-7 ff: !:. 1. :. .t. ::. :: , ;f .-: s;. -;!!,- -j!:-: .: 1. :I5; '!..... .. ..: ? ... !:...-.:;41:.'.: % ..

-, t1l. .7:;4:.. :..:,., ...::f...: .-- "'." -. :, .- a R-..If:llf-: ., ... :1..s:-::: -.. ..:: !:....... ., :.<;T :,:, .:!:,:.,f.1;.,..:: -:- -.i..-....' .:f....: .. ,:;1::;i, 't-.:.. ,-1-- f.V:f .i:.:,. :_ ;::.f ,f. .!:. ";":",. -.:;..:, .. :: ;. , .:,If;.-......: E ties .; ::,::,:Ai :.f"!,i1:::i,:.: ." .:.. .1. .,.;;: --. -. .f: .:,, ". i:,_i ;- ..'.- .. :.:,. 'I i.. t- 111y..., -t :-

f- .:. 1.f' %-;::f-. :, ." ..f---;,.,:-.- ..... f, .., .: I..': .-::. .:: ::,,.., 'Li: :s .:-- :,. 1., '::::,= .! ..", - ;.. , .:: :., -4U;;.; ,:.:, _-;,-;,::, ... .., ::.-.,:,-'-.',..`,: :: !:. '.: ;'Ill ':,i..:.; ''. - ,::.-,.- ..... ._ 1.1.f-- ... ....... .,. :.. ....., ....... 1, .. -17--!Pw.:. .::, ...

::

:.. ":.:: ....;: - .. ... "." -. ., i-. ;,...:-.. - '-N.': .I ut; ,4. :- .'. .: ::: .:.

..:: :. .: :.. .V 7 .! :s .,... ..... ., :'. ....... :- :- In't .. .1 1. ': f,1 .':'. !,.: ..:. -.,

.. .:.. .'.. :-: .. ,.,: .: ,.i.. .: ., 1... 1. :.,t.., 1:..... .. -.i.:, .:-- !I .-,,.;i s.''.... :... ,.: ........ v- .I: ,M.,. :!:: -.40 .... :1. ::.:..: .1 :: 4.:. :: ..:

'.. ". .. ::::.f, 1. ..` ]:.....:, .: .:: -::: '-: :- .. f.1I...:i,.. -..; ..: !..f,.: .. 1. :-. f:..,. :: .:. :;..::..%:, ..:..,: ...; :... ... -.': .:f :. ::..- .:::..,!f;.:.- :.:.:..: .::.. :: :, ,:-f1:;:,!,4-,:!.;-:, :, ,.: :.... ... .,.,.:,:.;, ..::.i:f :. 1.3: R .i.:;,-.:.:. ::,:.-... ': ".... .".'..;....'.111 ... f. !:::. ,.. .:.::.1fff,:,:, .. .:.:-:.. L:,... .:,.. :. 1",:,,:,:" .: ..:.-::::;:. ;. .: , .: .1It:s` ..V .1,mf,':n..,:, .,..... :,.-.-;:. ;.I.;,. :...... ........!:'s,:IN: :f;,;,t 1. .. ;:,-;,t:-;:A. , ,,..,;,:sM ::::::,:!: '. ..:: -::!::. ..;.:.;!. ..:. 1.:,V=,.:,;.. ": .gt

.. .;..I..11.."' -4,.:-.f,:;, '.:..: I..:,. ,--.-::..W.,.Vf ,,: ...'..:.. .:. :-.,:.f--W ,s.::: .,.::,. .:,..f"z:; -:f,.: .I:- :.:- ---. ................... .,.,:- :--7ff,:,. :.

... .:.!::.:n::.'f.'!..-... :... I.-ii ,, ::u-.:,s1xs;ut:.-:':1 ::. ::4. .. .f ;L .m...,.,::, .-:.i .4.:..,nf...:..-;.ii...i;t:..:.i:,...l.,.;:.:'i.I :.1:.:._ ..;: ..f .4s ::: ...1.:"M;:,,;: .::,.--- ..:::4: :: <% :1: .If n.: .:.4 .-..:.4:. .... f.--,!W !-s 1:-4. .. iA ;. :.:;'W =::-1 ..:.- -. ... ....:i -:::.f -4::::.--., ;::: ..... ::..:;:..'1t.:-;1.:M :f::: .'.'.:::n -::.-, .1-:: .::. .'.:. .. :: ::f ::.f. .I.1::-i;:,4f,:..;::.: .. .:,i: ..f ::f ff !.::s:. :j:.:--;! :.. ,-,.....% WU .: :.:f, 1: .i ..:. .Is:: -1tX1';f..:. 1, ..... ,., r,::, ::: ...... :u ,.. .I.,:., :-. .:.::: ::-:.

.:..;: .. 'II ...::. .;,!. :. .... .IE '. .:.N ..:.,::: .':fs,=h-.f,:'1% j.. .. .... ::f,;.;:n :;.,., il..,.

t.. N.,: ::., .I. ::.:. :. .... .. ..-::-:, :: .::. ......... '-: -...!..:' fs ..:: :. .. , iil...- --. :- -f- :f'- :- :- .. -. :1.::..:,::- :., :, ':.-,'. :f,..,:u .::f v .., ....Is;:.:-Ii. i. ...)s ... .:: :;::': :, ',,'if, :.::. ::! :::::-:,.;.:: ,:,.;! :V .z;.f- :-':: i::" .-: ...... .. ;A.f.: :.

...N .. -f :.:f'-.. , :, :,... I,. ...... :-.;z. :-:.h .: '... v,,:;.:4 ;: .,: ::..'!'W :..":-.,.-! ::. .,:. .f, --::.:

.:.,. .. ;, .; i..:` ,-, ::.. :.',..... ,;V .:..'; ..- -f .. .:..

.1.. .: :.. .; -., n" s,.1-...,. ..,. :t. !...::.;,. ... :. :: ., ....f.::. :, 1... ::i " ,7%.,. .,. .::- .is. ?..,,4. ..- -..: .:1 ..,..I,1:.-:.,:

-i :, :. '' - ... .:.:- I. i..: i.. :-,I:t.:If.. '.. .::

:. .1 .11- z, ;1, !':.:,. .. ::..:. -.., .::...= 'f'

:. 1, '!-. .. :.1. ". N -:-,-:.:-: ": :-- -:- :" -.......... .:. .....,I.......1i

.:

..i .. .: ..:- :.: :: .: .:... :. -f ". ,:.. .. :.:-!. ..: ... :.' ...: ...... '.. W.- .U.,:-. lu: ,..., `%...,; '. ....

.. .: .; , :: -.--;- ..,:;. fft:."'. ... ........ ....... ..----, :::.:,... -:,P".:,. ..." ---- !.--: .........'s. :,.;.::.:.: .i

:. .. ,:W .: .f: 2 .-.' '.: !:. .: - ,,, .,,. !:.-.. :: ,, ::..:.:"- :. ..: ...::.A "I ":. 1.1. :...-f !:. .: ::; ],. ,- .i .:.' -..-":.:: ,;.: .: '. ;I 4,:.,M :: .:... .. ,:. .: ,....., :. .".: ,, t, :1:-." -;. -.zn " ..:I.f- :.. -:.... ;; .:;. .. '::' .'.. :.. Mt., ..f..:-.. I.- -.

Proc. Nati. Acad. Sci. USA 91 (1994) 3193

A 80NGF + FK506

a60

~40'20 NGF

0 0.1 1 10 100

NGF, ng/ml

FIG. 3. Effects of FK506 on neurite outgrowth in PC12 cells.Cultures were treated with various concentrations of NGF in thepresence (c) or absence (U) of 100 nM FK506, and neurite sproutingwas measured at 48 hr. Outgrowth was quantitated by counting cellswith neuritic processes longer than 5 pmn. n = 4 separate experimentsfor each point and error bars represent SEM.

1-2 nM FK506, indicating that the binding sites correspondedto authentic FKBPs. PH]FK506 binding increased markedlyfollowing NGF treatment. Significant increases were evidentby 10-15 hr. Binding tripled by 20 hr and a modest furtherincrease was evident at 100 hr.FK506 and Rapanycin Increa Neurite EXtensiOn in PC12

Cells. As observed by others (19, 20), NGF potently stimu-lated neurite outgrowth with half-maximal stimulation at 1ng/ml and maximal augmentation at 50-100 ng/ml (Figs. 2and 3). FK506 (100 nM) markedly augmented the effect ofNGF by increasing sensitivity to NGF. Thus, FK506 reducedby a factor of 20-50 the NGF concentration needed to elicitmaximal outgrowth. Half-maximal outgrowth in the absenceof FK506 occurred with NGF at 5 ng/ml and in the presenceof FK506, with NGF at 0.1 ng/ml. With NGF at 10-100ng/ml, FK506 failed to produce additional neurite outgrowth.FK506 was extremely potent in its neurotrophic effects.

With a submaximal concentration ofNGF (1 ng/ml), FK506at 1 nM elicited the same maximal outgrowth observed withNGF at 50 ng/ml (Fig. 4). Half-maximal effects of FK506occurred at =0.1 nM. In the absence of NGF, FK506 failedto elicit neurite outgrowth (Fig. 3).Rapamycin is a potent immunosuppressant which is not

thought to act through calcineurin but which may influenceother phosphorylation cascades (21-28). Rapamycin potently

0 1 10 100 1000

FK506. pMNGF

(5 ) ny nl i)

FIG. 4. Concentration-response relationship for FK506 potenti-ation of neurite outgrowth in PC12 cells. Cells were treated for 48 hrwith NGF (1 ng/ml) and various concentrations of FK506. Neuriteoutgrowth response was measured as in Fig. 3. n = 4 separateexperiments for each data point. Stars indicate P < 0.001 byStudent's t test.

FIG. 5. [3H]FK506 autoradiography on dorsal root ganglionexplant cultures. After 26 days of culture with NGF (100 ng/ml), theextensive processes displayed abundant FKBP-associated silvergrains. Autoradiographic grains were abolished with 1 1&M unlabeledFK506.

blocks actions of FK506 that occur through FKBPs andcalcineurin, presumably by acting as an FK506 antagonist atFKBPs (7, 21, 29, 30). Rapamycin (1 juM) failed to block theneurotrophic actions of FK506 (data not shown). Instead,rapamycin was itself neurotrophic, providing major neuriteoutgrowth at 1 nM. Rapamycin and FK506 seemed to act viadifferent mechanisms. Thus, rapamycin augmented the num-ber of processes as well as their length, whereas FK506primarily increased neurite length. Moreover, effects ofFK506 and rapamycin appeared to be additive (data notshown).FK506 Is Neurotrophic for Sensory Ganglia. We examined

actions of FK506 on primary cultures of dorsal root gangliafrom rats at embryonic day 16. Autoradiography ofPH]FK506 binding sites revealed substantial levels ofFKBP-associated silver grains in these ganglia (Fig. 5). At 1 gMunlabeled FK506, autoradiographic grains were abolished,indicating the specificity of binding. As reported previously(31), NGF (100 ng/ml) markedly increased the number andlength of ganglion processes (Fig. 6). FK506 (1 AuM) aloneproduced a similar neurotrophic effect, while as little as 1 nMFK506 produced a noticeable increase in growth. Rapamycin(1 1LM), which acts as an FK506 antagonist, completelyblocked the effects of FK506 (1 ,uM); thus the action ofFK506 displays a drug specificity characteristic ofFKBP (3,9, 10, 32).Whereas FK506 failed to stimulate neurite outgrowth in

PC12 cells in the absence of added NGF, in sensory gangliaFK506 alone was neurotrophic. Schwann cells in the gangliacan fabricate NGF, and the production of NGF by Schwanncells is regulated by protein phosphorylation (33). To ascer-tain whether the actions of FK506 alone involved potentia-tion of endogenous NGF, we examined the influence ofantibodies to NGF (Fig. 6). Anti-NGF markedly reduced theneurotrophic effects ofFK506 (1 lxM). The anti-NGF was notacting in a toxic fashion, as we observed no morphologicevidence of toxicity in the cells exposed to anti-NGF in thepresence or absence of added NGF.FK506 was extremely potent in stimulating neurite out-

growth. As little as 1 pM FK506 produced detectable aug-mentation. Progressively greater outgrowth occurred at 0.1and 10 nM FK506 (data not shown), whereas maximaloutgrowth required 1 I&M FK506.The time course of neurite outgrowth was similar at all

concentrations of NGF and FK506. Some outgrowth wasevident by 1 day, and growth began to plateau at 5-6 days.

Neurobiology: Lyons et al.

Proc. Nati. Acad. Sci. USA 91 (1994)

i

FIG. 6. Phase-contrast micrographs of dorsal root ganglia grown with NGF at 100 ng/ml (A), FK506 at 1 1AM (B), FK506 at 1 IM plusanti-NGF antibody (C), no added growth factor (D), FK506 at 1 pM (E), or at FK506 at 1 ,AM plus rapamycin at 1 pM (F). NGF producedabundant axon outgrowth (A), as did 1 AM FK506 (B). The effects of FK506 were substantially decreased by reducing the concentration to 1pM (E). However, neurite outgrowth with 1 pM FK506 was greater than in its absence (D). FK506 effects were also diminished by addinganti-NGF antibody to eliminate the effects ofNGF produced by nonneuronal cells in the cultures. The abundant neurites that occurred in largefascicles in response to NGF (100 ng/ml) (A) or 1 pM FK506 (B) appear white, whereas small fascicles or individual neurites appear black.Nonneuronal cells, Schwann cells, and some fibroblasts are more evident with 1 pM FK506 (E) or anti-NGF antibody (C) than with 1 pM FK506(B). NGF produced by nonneuronal cells in the cultures resulted in the limited axon outgrowth seen in cultures with no added growth factors(D). The large number ofrefractile nonneuronal cells, appearing white, tend to overshadow the few neurites (D). Rapamycin completely inhibitedaxon outgrowth in the presence of FK506 (F). Micrographs are representative of 12-30 ganglia from each experimental condition. Differencesbetween all experimental groups were highly reproducible. (Bar = 250 pm.)

DISCUSSIONThe major finding of this study is the dramatic neurotrophicaction of FK506 in PC12 cells and sensory ganglia and ofrapamycin in PC12 cells. These actions are extraordinary interms of potency, with as little as 1 pM FK506 beingneurotrophic. Physiologic relevance is likely, as similar neu-

rotrophic actions occur in two distinct cellular systems.Moreover, a preliminary report describes enhanced sciaticnerve growth in intact rats after axotomy (34).FK506 neurotrophic effects appear to involve FKBPs in

sensory ganglia, since the effects of FK506 were reversed bylow concentrations of rapamycin, a known antagonist ofFK506 at FKBPs (7, 21, 29, 30). The failure of rapamycin toblock FK506 effects in PC12 cells probably reflects theseparate stimulatory effects of rapamycin. Mechanisms forrapamycin stimulation of neurite outgrowth in PC12 cells arenot immediately evident. Its immunosuppressant actions are

thought to involve mechanisms different from those ofFK506. Rapamycin can inhibit S6 kinase, which phosphory-lates the S6 protein ofthe small ribosomal subunit (22-24, 26,28). Rapamycin also inhibits phosphatidylinositol 3-kinase(25).

It is tempting to speculate that FK506 acting throughFKBP-12 inhibits calcineurin to increase levels of phosphor-ylated calcineurin substrates. Such a mechanism would fitwith findings that neurite outgrowth in several systems isassociated with phosphorylation of numerous proteins in theneurite extensions (35, 36). Protein kinase C-mediated phos-phorylation has been implicated in process outgrowth duringneuronal regeneration (37-41). Other evidence suggests in-hibitory effects of protein kinase C in neuronal processextension (42-45).GAP43 is a prominent calcineurin substrate that is highly

concentrated in neurites (14), and its phosphorylation isregulated by FKBP (4). GAP43 may not be directly involved

3194 Neurobiology: Lyons et al.

Proc. Natl. Acad. Sci. USA 91 (1994) 3195

in neurite extension, as PC12 cell lines with low levels ofGAP43 display normal neurite outgrowth (46). However,GAP43 and its phosphorylation may be involved in targetingneurites, as levels of phosphorylated GAP43 are increasedwhen neurites approach their targets (47). Phosphorylation ofGAP43 may also influence mobilization ofCa2+ that regulatesneurite extension. Phosphorylated GAP43 inhibits formationof phosphatidylinositol bisphosphate, which should diminishlevels of inositol 1,4,5-trisphosphate and associated Ca2+release (17). In addition, phosphorylation of GAP43 de-creases its affinity for calmodulin, with the resultant freecalmodulin available to bind Ca2+ (17).Immunophilins may act at sites besides calcineurin which

affect Ca2+ that regulates neurite outgrowth. FKBP binds tothe ryanodine receptor, a Ca2+ release channel (48). Inskeletal muscle sarcoplasmic reticulum, FK506 dissociatesFKBPs from the ryanodine receptor to facilitate the Ca+-induced Ca2+ release mechanism (49). hI addition, FK506acts at other sites, including FKBP-25 (50, 51), steroidreceptors (52, 53), and other, unidentified targets such asthose related to FKBP13 (54). Thus, other mechanisms mayplay some role in neurite extension.These neurotrophic actions may have therapeutic ramifi-

cations. Numerous neurotrophic proteins, such as brain-derived neurotrophic factor, ciliary neurotrophic factor, andNGF are being clinically evaluated in numerous diseasestates (55). Difficulties in manufacture of these large proteinsand delivery to target sites provide serious barriers to ther-apeutic application. The extreme potency of FK506 is in therange of that for neurotrophic proteins. Drugs such as FK506are readily synthesized and can cross the blood-brain barrier.Thus, besides potential therapeutic effects for neuroprotec-tion in conditions such as stroke (10), FK506 and other smallmolecules that interact with immunophilins may be of use infacilitating neuronal repair.

We thank Fujisawa Pharmaceutical (Osaka) for FK506, Dr. S.Sehgal at Wyeth-Ayerst for rapamycin, and J. Mong for technicalassistance. This work was supported by U.S. Public Health ServiceGrant DA00266, Research Scientist Award DA00074 to S.H.S., agrant from the International Life Sciences Institute, and the W. M.Keck Foundation. T.M.D. is supported by grants from the AmericanAcademy of Neurology and by a U.S. Public Health Service ClinicalInvestigator Development Award (NS01578). J.P.S. is supported bya postdoctoral fellowship from the U.S. Public Health Service(MH10101). W.E.L. is supported by National Institute of Environ-mental Health Sciences Training Grant ES07141.

1. Handschumacher, R. E., Harding, M. W., Rice, J., Drugge, R. J. &Speicer, D. W. (1984) Science 226, 544-547.

2. Swanson, S. K.-H., Born, T., Zydowsky, L. D., Cho, H., Chang,H. Y., Walsh, C. T. & Rusnak, F. (1992) Proc. Natl. Acad. Sci.USA 89, 3741-3745.

3. McKeon, F. (1991) Cell G6, 823-826.4. Steiner, J. P., Dawson, T. M., Fotuhi, M., Glatt, C. E., Snowman,

A. M., Cohen, N. & Snyder, S. H. (1992) Nature (London) 358,584-587.

5. Liu, J., Farmer, J. D., Jr., Lane, W. S., Friedman, J., Weissman,I. & Schreiber, S. L. (1991) Cell66, 807-815.

6. Liu, J., Albers, M. W., Wandless, T. J., Luan, S., Alberg, D. G.,Belshaw, P. J., Cohen, P., MacKintosh, C., Klee, C. B. &Schreiber, S. L. (1992) Biochemistry 31, 3896-3901.

7. Dumont, F. J., Melino, M. R., Staruch, M. J., Koprak, S. L.,Fisher, P. A. & Sigal, N. H. (1990) J. Immunol. 144, 1418-1424.

8. Fruman, D. A., Klee, C. B., Bierer, B. E. & Burakoff, S. J. (1992)Proc. Nat!. Acad. Sci. USA 89, 3686-3690.

9. Schreiber, S. L. & Crabtree, G. R. (1992) Immunol. Today 13,136-142.

10. Dawson, T. M., Steiner, J. P., Dawson, V. L., Dinerman, J. L.,Uhl, G. R. & Snyder, S. H. (1993) Proc. Nat!. Acad. Sci. USA 90,9808-9812.

11. Bredt, D. S., Ferris, C. D. & Snyder, S. H. (1992) J. Biol. Chem.267, 10976-10981.

12. Dawson, V. L., Dawson, T. M., London, E. D., Bredt, D. S. &Snyder, S. H. (1991) Proc. Natl. Acad. Sci. USA 88, 6368-6371.

13. Dawson, V. L., Dawson, T. M., Bartley, D. A., Uhl, G. R. &Snyder, S. H. (1993) J. Neurosci. 13, 2651-2661.

14. Liu, Y. & Storm, D. R. (1989) J. Biol. Chem. 264, 12800-12804.15. Snipes, G. J., Costello, B., McGuire, C. B., Mayes, B. N., Bock,

S. S., Norden, J. J. & Freeman, J. A. (1987) Prog. Brain Res. 71,155-175.

16. Benowitz, L. I. & Routtenberg, A. (1987) Trends NeuroSci. 10,527-532.

17. Skene, J. H. P. (1989) Annu. Rev. Neurosci. 12, 127-156.18. Tetzlaff, W., Zwiers, H., Lederis, K., Cassar, L. & Bisby, M. A.

(1989) J. Neurosci. 9, 1303-1313.19. Greene, L. A. & Tischler, A. S. (1976) Proc. Nat!. Acad. Sci. USA

73, 2424-2428.20. Yanker, B. A., Benowitz, L. I., Villa-Komaroff, L. & Neve, R. L.

(1990) Mol. Brain Res. 71, 39-44.21. Bierer, B. E., Mattila, P. S., Standaert, R. F., Herzenberg, L. A.,

Burakoff, S. J., Crabtree, G. & Schreiber, S. L. (1990) Proc. Nat!.Acad. Sci. USA 87, 9231-9235.

22. Calvo, V., Crews, C. M., Vik, T. A. & Bierer, B. E. (1992) Proc.Nat!. Acad. Sci. USA 89, 7571-7575.

23. Chung, J., Kuo, C. J., Crabtree, G. R. & Blenis, J. (1992) Cell 69,1227-1236.

24. Kuo, C. J., Chung, J., Fiorentino, D. F., Flanagan, W. M., Blenis,J. & Crabtree, G. R. (1992) Nature (London) 358, 70-73.

25. Kunz, J., Henriquez, R., Schneider, U., Deuter-Reinhard, M.,Movva, N. R. & Hall, M. N. (1993) Cell 73, 585-596.

26. Price, D. J., Grove, J. R., Calvo, V., Avruch, J. & Bierer, B. E.(1993) Science 257, 973-977.

27. Jin, Y. J. & Burakoff, S. J. (1993) Proc. Natl. Acad. Sci. USA 90,7769-7773.

28. Ferrari, S., Pearson, R. B., Siegman, M., Kozma, S. C. & Thomas,G. (1993) J. Biol. Chem. 268, 16091-16094.

29. Schreiber, S. L. (1991) Science 253, 283-287.30. Dumont, F. J., Staruch, M. J., Koprak, S. L., Melino, M. R. &

Sigal, N. H. (1990) J. Immunol. 144, 251-258.31. Thoenen, H. & Barde, Y. A. (1980) Physiol. Rev. 60, 1284-1335.32. DeFranco, A. L. (1991) Nature (London) 352, 754-755.33. Matsuoka, I., Meyer, M. & Thoenen, H. (1991) J. Neurosci. 11,

3165-3177.34. Gold, B. G., Storm-Dickerson, T., Austin, D. R. & Katoh, K.

(1993) Soc. Neurosci. Abstr. 19, 1316.35. Levi, A., Biocca, A., Cattaneo, A. & Calissano, P. (1988) Mol.

Neurobiol. 2, 201-226.36. Fujita, K., Lazarovici, P. & Guroff, G. (1989) Environ. Health

Prosp. 80, 127-142.37. Mechta, S., Hsu, L., Jeng, A. Y. & Chen, K. Y. (1993) J. Neuro-

chem. 60, 972-981.38. Hsu, L. (1988) Anat. Embryol. 179, 511-578.39. Hall, F. L., Fernyhough, P., Ishir, D. N. & Vulliet, P. R. (1988) J.

Biol. Chem. 263, 4460-4466.40. Hashimoto, S. & Hagine, A. (1989) J. Neurochem. 1675, 1685.41. Bixby, J. L. (1989) Neuron 3, 287-297.42. Morrison, R. S., Gross, J. L. & Moskal, J. R. (1988) Brain Res. 473,

141-146.43. Mattson, M. P., Guthrie, P. B. & Kater, S. B. (1988) J. Neurosci.

Res. 21, 447-464.44. Girard, P. R. & Kuo, J. F. (1990) J. Neurochem. 54, 300-306.45. Reinhold, D. S. & Nect, K. E. (1989) J. Biol. Chem. 102, 821-829.46. Baetge, E. E. & Hammang, J. P. (1993) Neuron 6, 21-30.47. Meiri, K. F., Bickerstaff, L. E. & Schwob, J. E. (1991)J. Cell Biol.

112, 991-1005.48. Jayraman, T., Brillantes, A. M., Timerman, A. P., Fleischer, S.,

Erdjument-Bromage, H., Tempst, P. & Marks, A. (1992) J. Biol.Chem. 267, 9474-9477.

49. Timerman, A. P., Ogunbumni, E., Freund, E., Wiederrecht, G.,Marks, A. R. & Fleischer, S. (1993) J. Biol. Chem. 268, 22992-22999.

50. Galat, A., Lane, W. S., Standaert, R. F. & Schreiber, S. L. (1992)Biochemistry 31, 2427-2434.

51. Jin, Y. J., Burakoff, S. J. & Bierer, B. E. (1992) J. Biol. Chem. 267,10942-10945.

52. Tai, P.-K. K., Albers, M. W., Chang, H., Faber, L. E. &Schreiber, S. L. (1992) Science 256, 1315-1318.

53. Yem, A. W., Tomasselli, A. G., Heinrikson, R. L., Zurcher-Neely,H., Ruff, V. A., Johnson, R. A. & Deibel, M. R., Jr. (1992) J. Biol.Chem. 267, 2868-2871.

54. Jin, Y.-J., Albers, M. W., Lane, W. S., Bierer, B. E., Schreiber,S. L. & Burakoff, S. J. (1991) Proc. Natl. Acad. Sci. USA 88,6677-6681.

55. Phelps, C. H., Gage, F. H., Growden, J. H., Hefti, F., Harbaugh,R., Johnson, M. V., Khachaturian, Z. S. & Mobley, W. C. (1989)Neurobiol. Aging 10, 205-207.

Neurobiology: Lyons et al.