In situ degradation of rapidly transported proteins in nerve terminals of retinal ganglion cells

'Veto me ~t'pl~e Letters, 63 (1986) 221 224

22 I

Plsevler Sctentlfic Pubhshers Ireland Ltd

NSL 01731

IN S I T U D E G R A D A T I O N OF R A P I D L Y T R A N S P O R T E D P R O T E I N S IN NERVE T E R M I N A L S OF R E T I N A L G A N G L I O N CELLS

S GUSTAVSSON and J -O K A R L S S O N * Imtttute o~ Neurohtologt, Umter~ltt o/Gotehorg, P 0 Bo~ 33 031, S-400 33, Gotehorg 33 fan eden/ (Recelved June 24th. 1985, Re'.lsed "version received and accepted October 10th. 1985)

h~ i winds

axonal transport

calcium

protein

proteolysls

rabbit

The m SllU degradation of rapidly transported proteins In ner'.,e terminals of retinal ganglion cells 'aas stud~ed In prates nl the superior colhculus of the rabbit Proteolytlc degradatmn was fi)und to be dependent upon extracel[ular calcium and intact calcium channels Degradation was inhibited by leupeptm ,tnd SH-group blocking agents

It is well known that the entry of external Ca 2+ through the plasma m e m b r a n e is the primary s,gnal for neurotransmltter release [1] Th~s increase m nerve terminal Ca e ~ levels m a y also regulate the act~,,qty of ('aactivated proteolytlc enzymes A pair of Ca-activated neutral proteases has recently been described m the cytosol o f brain and other organs [7] These enzymes are active ,tt micro- and mllhmolar concentrations of Ca 2+ (calpam I and II, respectively) In v~vo studies suggest that the half-hfe for prote,ns of rapid axonal transport m the nerve termmals Is considerably shorter than that o f slowly transported proteins At least some c o m p o n e n t s appear to have half-hves in the order o f a l'e,a, hours [2. 6, 11] Proteolyttc degradation o f rapidly transported proteins m homogenates o f nerve tissue 1-ms earher been characterized [3, 4, 10] The characteristics o f proteol.~tm degradation o f slowly transported c o m p o n e n t s have been descmbed in relatlvel? mtact systems IS, 9] Reports c o n c e r m n g the c o n d l h o n s for proteolytlc degradation o f rapidly transported proteins in relatively intact systems are scarce Early expemments on a superfusmn %'stem c o n t a l n m g shces o f the supemor colhculus from the rabbit demonstrated that chemical depolarlzatzon of the t~ssue with high K evoked a release of trlchloroacetlc acid (TCA)-soluble radmactlwty derived from rapidly transported proteins [10] This evoked release was dependent upon extracellular Ca Kanje et al [4] have recently described proteolyttc degradation o f rapidly transported proteins m desheathed frog scmtlC nerves exposed to 5 or 50 m m Ca, whmh was mcreased following apphcatlon o f a Ca m n o p h o r e The present stud3, was designed, m a rela-

* &uthor for correspondence

[l~[M-t940 86 $ 03 50 ,e, 1986 Elsevier Sclenlflic Publishers Ireland Lid

222

ttvely mtact system, to further eluctdate the role of Ca-activated neutral thlol proteases tn nerve terminals Rabbtts of both sexes, wetghing ca 2 kg, were injected mtraocularly w~th 50 ~C~ of L-[5-3H]proline (26 Ci/mmol, The Radiochemtcal Centre, Amersham) into both eyes Eighteen to 24 h followmg the tsotope mjecttons, the animals were killed by an air embolus and perfused with 150 mM NaC! in 10 m M Trts-HCI (pH 7 2) The superxor colliculus was rapidly dissected out, and the superficial 2 mm layer was divided mto 4 pxeces of equal size. The pieces were mcubated m 1 ml 'complete buffer' for 60 m m at 39"C m shaking water bath. The complete buffer had the following composition (in m M ) 100 NaCI, 5 KCI, 5 glucose, I proline, 2 Ca, 1 Mg and 50 TrIs-HCI (pH 7 2) Following incubation the samples were homogenized with 10",, TCA and centrifuged The TCA precipitate was dissolved in 1 M N a O H Ahquots from the TCA supernatant and from the solubihzed TCA precipitate were taken for liquid scintillation counting Data are expressed as TCA-soluble dpm/total dpm × 100 An increase m thts ratio was considered to represent proteolytlc degradation Zero ttme values were subtracted from presented data Proteolyt~c degradation of raptdly transported protems from retinal ganghon cells was hnear for at least 3 h in pieces of the supertor colliculus (Fig. l) During the per~-

15-

r~

< 1 =: 10~ 0 I,M ¢z Iz I.iJ 00: I,U o. 5.

0

•

5'0

•

100 150 I N C U B A T I O N T I M E (min)

200

Fig 1 Proteolysls m pieces of the superior colliculus Radloactzvlty in the TCA-soluble fraction as percentage of the total ra&oactiwty plotted as a function of incubation Ume Average of two experiments

223 TABLE I EFFECTS OF DIFFERENT COMPOSITION OF THE INCUBATION MEDIA ON PROTEOL~'TIC DFGRADATION l)egradatlon ol" axonally transported proteins during I h incubation m complete buffer is referred to a,, Ill()",, A,,erage values (-+ S D n 3 5) are expressed as percentage of the value obtained using t.ompletc buffer Percent degradation ( -+S D ) (omplcte huller Ca ~ Mg" +EDTA (5 raM) + ~'erapamll(0 1 raM) + Leupeptm (100 g'ml) + I1( MB (0 I raM) + HCMB (1 inM)

100+ 4 45 _4_13 98 _+14 25+ 7 78+ 8 5~+ 15 83+ 19 67 + 14

od studied, a p p r o x i m a t e l y 17°o of the labelled protems were b r o k e n d o w n (Fig I) D e g r a d a t i o n In a h o m o g e n a t e system has been shown to be much lower and hnear for only a p p r o x i m a t e l y 5 m m [3] D e g r a d a t i o n was clearly d e p e n d e n t u p o n extracellular calcmm ions R e m o v a l of Ca or addition of e t h y l e n e d l a m m e t e t r a a c e t l c acid ( E D T A ) to the m e d i u m decreased d e g r a d a t i o n to 45 and 25",,, respectively, of the n o r m a l level (Table I) R e m o v a l of m a g n e s i u m had no effect (Table I) A d d i t i o n of the C a - c h a n n e l blocking drug, verapamll, caused a decreased d e g r a d a t i o n Leupeptm and the S H - g r o u p blocking agent p - h y d r o x y m e r c u r l b e n z o a t e ( H C M B ) . were both effective m decreasing the proteolbtic act~\lt? (Table I) The experimental system used m a y in several ways mimic the in wvo e n v i r o n m e n t of the nerve terminals However, c o m p h c a t m g factors due to the separation of the nerve terminal region from the axons may arise_ N o t h i n g ts k n o w n a b o u t the electrophysiological activity of the tissue d u r i n g the time period studied Most of the retinal afl'erents to the supemor colhculus terminate very close to the colhcular surface (stratum grlseum superficmle)_ The diffusion d~stance between the m e d i u m a n d most of the nerve terminals of optic o n g m is p r o b a b l y less than 100 ltm [12] The two mhtbitops of the Ca-activated neutral thlol proteases used were able to affect the actlwty of the enzyme(s) Leupeptm, which is a relatively h y d r o p h o b i c molecule, was particularly effective as an i n h i b i t o r In a system with httle diffusion barriers, such as tn a homogenate, the sulfhydryl blocker, H C M B , was much more effectave than leupeptm [3] The calcium-reduced s t i m u l a t i o n of proteolytic activity was p r o b a b l y medmted via the C a - c h a n n e l , as evidenced by the effect of verapamll The l n t r a t e r m m a l concentratton of Ca in the present experimental system is not k n o w n We have shov+n that the r m c r o m o l a r form o f calpam (calpam I), isolated from r a b b i t brain m an in vitro assay, ts capable o f d e g r a d i n g rapidly t r a n s p o r t e d proteins in nerve terminals of optic omgm at m~cromolar c o n c e n t r a t i o n s of Ca +++ [5] The m a r k e d effect of extra-

224 c e l l u l a r c a l c i u m levels o n p r o t e o l y t i c d e g r a d a t t o n m n e r v e t e r m i n a l s d u r i n g in sttu m c u b a t t o n m a y c o r r e s p o n d to t h e in VlVO s i t u a t i o n . I n thts c a s e d e p o l a r i z a t i o n o f t h e p r e s y n a p t ~ c m e m b r a n e , w~th a n i n c r e a s e d influx o f C a , m a y r e g u l a t e i n t r a t e r m l hal p r o t e o l y t t c act~vtty T h i s w o r k w a s s u p p o r t e d by t h e S w e d t s h M e d t c a l R e s e a r c h C o u n c i l ( N o . 5932), T o r s t e n a n d R a g n a r S o d e r b e r g s s t i f t e l s e r a n d M a g n . B e r g v a l l s sttftelser_ W e t h a n k I n g e r J a n s o n f o r e x c e l l e n t t e c h n t c a l a s s i s t a n c e a n d G u l l G r 6 n s t e d t f o r c a r e f u l secretartal w o r k 1 Erulkar. S D and Fine, A , Calcium In the nervous system, Rev Neuroscl, 4 (1979) 179 232 2 Goodrum, J F , Toews, A D and Morell, P , Axonal transport and metabohsm of [aH]fucose and [~sS]sulfate-labeled rnacromolecules m the rat visual system, Brain Res, 176 (1979) 25_%272 3 Gustavsson, S and Karlsson, J -O, Release of acid-soluble radloactlwty from rapidly transported proteins in homogenates of rabbit supertor colhculus, Neurochem Int. 7 (1985) 639-645 4 Kanje, M , Lazarewlcz, J Ekstrom. P and Edstrom, A , Ca 2+- activated protease activity m frog sciatic nerve characterization and effect on rapidly transported axonal proteins, Brain Res, 327 (1985) 29 36 5 Karlsson, J -O, Gustavsson, R , Hall, C and Ndsson, E, A simple one-step procedure lbr the separation of calpam I, calpam II and calpastatm, Biochem J_, 231 (1985) 201-204 6 Karlsson, J -O and Sjostrand, J , Synthesis, migration and turnover of protein m retinal ganglion cells. J Neurochem, 18 (1971) 749 767 7 Murachl, T , Calpaln and calpastatm, TIBS, 8 (1983) 167-169 8 Nlxon, R A , Protein degradation in the mouse visual system I Degradation of axonally transported and retinal proteins, Brain Res, 200 (1980) 69 83 9 Sandberg, M , Hamberger, A , Karlsson, J -O and Tlrllhm, B, Potassmm-sttmulated release of axonally transported radioactivity from slices of rabbit superior colhculus, Brain Res 188 (1980) 175 183 l0 Sandberg, M , Hamberger, A , Jacobson, 1 and Karlsson, J -O The role ofcalcmm ions in the formation and release of small molecular weight substances from optic nerve terminals, Neurochem Res 5 (1980) 1185- 1198 11 Wlllard, M , Cowan, W M and Vagelos, P R , The polypeptide composition of mtra-axonally transported proteins evidence for four transport velocities, Proc Natl Acad Scl USA, 71 (1974) 2183 2187 12 Vrensen, G and de Groot, D , Quantitative aspects of the synaptic orgamzatlon of the superior colhculus m control and dark-reared rabbits, Brain Res, 134 (1977) 417~428 ,

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