N~wron,

Vol. 6, 635-647,

April,

1991, Copyright

0 1991 by Cell Press

Expression of the Growth-Associated Protein GAP-43 in Adult Rat Retinal Ganglion Cells following Axon Injury S. Kathleen Doster,** Andres M. Lozano,+§ Albert J. Aguayo,+ and Mark B. Willard* *Department of Anatomy and Neurobiology Washington University School of Medicine St. Louis, Missouri 63110 +Neurosciences Unit Montreal General Hospital and McGill University Montreal, Quebec Canada H3G IA4

Summary We have studied the expression of the growth-associated protein GAP-43 after injury to theaxons of adult rat retinal ganglion cells (CNS neurons that do not normally regenerate injured axons). Both the biosynthetic labeling of GAP-43 and the GAP-43 immunoreactivity of the retina increased after axotomy, but only when the injury was within 3 mm of the eye. These results suggest the following conclusions: First, axon injury is sufficient to alter CAP-43 expression in CNS neurons, even in the absence of regeneration. Second, mechanisms that regulate GAP-43 expression are sensitive to the length of uninterrupted axon remaining after injury. Finally, the conditions that favor increased GAP-43 are similar to those that favor regrowth of injured CNS axons into grafts of peripheral nerve, suggesting that GAP-43 induction is accompanied by an increased potential of injured CNS neurons to regenerate. introduction Among the metabolic changes that often accompany the extension of axons by neurons is their increased svnthesis and accumulation of certain proteins designated growth-associated proteins (GAPS) (reviewed in Skene, 1989; Willard et al., 1985). One of these proteins, GAP-43, undergoes rapid axonal transport (Skene and Willard, 1981a, 1981b) and is a major component of the membranes of growth cones (DeGraan et al., 1985; Meiri et al., 1986; Skene et al., 1986). In addition, it is a component of certain presynaptic terminals (Gispen et al., 1985a; Benowitz et al., 1988; McGuire et al., 1988); its phosphorylation by protein kinase C has been reported to be associated with long-term potentiation in the hip pocampus (Nelson and Routtenberg, 1985; Benowitz and Routtenberg, 1988) and with release of neurotransmitter (Dekker et al., 1989). Although the precise function of GAP-43 is not known, certain properties have suggested that it may influence second messen* Present address: Department of Neurosurgery, University of Minnesota School of Medicine, Minneapolis, Minnesota 55455. 5 Present address: Toronto Western Hospital, 399 Bathurst, Toronto M5T 250, Canada.

ger systems. For example, it is reported to bind to calmodulin in a Ca2+-sensitive manner (Andreasen et al., 1983; Cimler et al., 1987), to influence phosphoinositide metabolism (Gispen et al., 1985b), and to stimulate hydrolysis of GTP by the G protein Go (Strittmatter et al., 1990). GAP-43 is identical to proteins designated Fl (Nelson and Routtenberg, 1985), B50 (Zwiers et al., 1976), PP46 (Katz et al., 1985), and P57 or neuromodulin (Andreasen et al., 1983). A relationship between GAP-43 and axon growth is suggested by observations that there is more GAP-43 in certain neurons during development than in the adult and that synthesis of GAP-43 increases when certain adult neurons regenerate injured axons (Jones and Aguayo, 1990, Sot. Neurosci., abstract; Biffo et al., 1990; Skene and Willard, 1981a, 1981b; Benowitz and Lewis, 1983; Grafstein, 1987; Perry et al., 1987; Moya et al., 1988; Kalil and Skene, 1986). This relationship suggests that the expression of GAP-43 may respond to the same signals that regulate axon growth. The central nervous system (CNS) of adult mammals provides an experimental system for examining the relationship between altered GAP-43expression and axon regeneration. When neurons are injured, they do not normally regenerate their axons within the CNS; however, extensive studies have shown that injured CNS neurons will regenerate axons into segments of peripheral nerve that have been grafted into the CNS (for review, see Aguayo, 1985; Aguayo et al., 1990). The regeneration of these axons into the grafts depends upon the position of the injury; only axons injured within a few millimeters of their cell bodies are regenerated (David and Aguayo, 1981; Benfey and Aguayo, 1982; Richardson et al., 1984; Vidal-Sanz et al., 1987). These considerations raise the question of whether the metabolic changes manifested by induction of GAP-43 occur during regeneration of CNS neurons and if so, whether they are signaled by axotomy or alternatively, whether they require additional signals supplied bythe peripheral nerve grafts. Here we have investigated the response of GAP-43 in rat retinal ganglion cells to injury at various positions along their axons in the optic nerve. We observe that both GAP-43 immunoreactivity of whole mounts of the retina and axonal transport of GAP-43 in the optic nerve respond to axotomy in the absence of regeneration, but only when the axotomy is close to the eye; the dependence upon position is similar to the dependence previously established for regeneration of these axons into peripheral nerve grafts. Results Evaluation of Anti-GAP-43 Binding to Retinal Whole Mounts as an Assay for Changes in GAP-43 One preparation of affinity-purified anti-GAP43 antibody, produced in rabbits against rat GAP-43 as de-

Neuron 636

abed Figure

1. Specificity

of Anti-GAP-43

e

1990, Sot. Neurosci., abstract). Figure 2 shows that the axon bundles of the antibody-labeled retina of l-day-old rats (n = 4) fluoresced brightly (Figures 2a and 2b); on the other hand, the retinas of IO-day-old rats (n = 5) were much less reactive (Figures 2d and 2e). In adult rats (n = 27), no antibody-specific fluorescence was detected (Figures 2g and 2h), with the rare exception of a labeled cell body. In individual retinal axons of the l-day-old animal, the fluorescence was punctate and discontinuous along the axon. The cell bodies were not stained. The decrease in staining of the older animals was apparently not due to decreased access of the antibody, because the staining with an anti-neurofilament antibody did not decrease in older animals (data not shown). These experiments show that fluorescent antibody staining of wholemounted retinas under these conditions can readily detect GAP-43 in the retinal axons of l-day-old animals, but is not sufficiently sensitive to detect the levels of GAP-43 in the adult retina. We used this procedure to investigate whether changes in the expression of GAP-43 of similar magnitude occur following axotomy of retinal ganglion cells in the adult.

Antibody

Samples of a homogenate of brain (from a l-day-old rat) containing60Bg (lanea), 150 ug (lane b), and 600 ug (lanec) of protein were electrophoresed, and the proteins were transferred to nitrocellulose paper. The affinity-purified anti-CAP-43 antibody (2.8 ug/ml) bound only to material with the same electrophoretic mobility as purified GAP-43 (lane d). Antibody binding was detected by reaction of horseradish peroxidase with diaminobenzidine (Experimental Procedures). Lane e is identical to lane c, except that preimmunization IgG (5.6 Kg/ml) was substituted for anti-GAP-43.

scribed in Experimental Procedures, was used in all of the experiments reported here. Its specificity for GAP-43 is indicated by the following observations: First, it bound to an affinity column containing purified GAP-43. Second, it bound to purified GAP-43 on Western blots (Figure 1, lane d). Third, it bound to a single band with the same electrophoretic mobility as purified GAP-43on Western blots of neonatal rat brain protein that had been dissolved in SDS and separated by SDS-PAGE (Figure l, lanesa-c). Fourth, on Western blots of neonatal rat brain proteins separated by twodimensional gel electrophoresis (IEF-SDS-PAGE) the antibody bound to several horizontally arrayed acidic spots, or a streak, whose mobility in the SDS-PAGE dimension relative to standard proteins depended upon the acrylamide concentration in the unusual manner that is characteristic of GAP-43 (Jacobson et al., 1986; Benowitz et al., 1987; data not shown). To determine whether the binding of anti-GAP-43 antibodies to whole mounts of rat retina is sensitive to changes in the expression of GAP-43, we assessed its ability to detect the decrease (more than 20-fold) in GAP-43 that occurs during postnatal development of mammalian retinal ganglion cells (Skene and Willard, 1981b; Freeman et al., 1986; Jones and Aguayo,

GAP-43 lmmunoreactivity following Axotomy To investigate the effect of axotomy upon expression of GAP-43 in retinal ganglion cells, we stained whole mounts of rat retina at various times after cutting or crushing the optic nerve of adult rats. When the optic nerve was cut intracranially at distances from 6-10 mm from the globe (n = 14 animals), we observed no increase in immunoreactivityof retinal whole mounts at any time in the following 30 days (Figure 3). On the other hand, when the optic nerves were cut or crushed intraorbitally l-3 mm from the globe (n = 31 animals), the labeling of the retinal whole mountswith anti-GAP-43 antibody increased dramatically, beginning 6 days after the axotomy. The reactivity of the retinas appeared to reach maximum levels at 8-12 days; after 20days, it had diminished significantly, and by 25 days it was indistinguishable from the labeling of retinas that had not been axotomized (Figure 3). Figure 4 compares a retina 12 days after axotomy (a) with the retina of an unaxotomized control (b). The labeled axons extended to the periphery of the retina, 3-4 mm from the optic disc, and the appearance of the immunofluorescencewithin theaxonswas similar to that of the l-day-old rat (Figures 2a and 2b); the labeling was discontinuous and punctate along the length of the axons (Figures 5a and 5~). There was no difference in the intensity of the reaction IO days after injury between animals whose optic nerves had been cut, as compared with crushed. To determine whether the increase in immunoreactivitywas a consequence of axotomy, rather than nonspecific aspects of the surgical procedure (such as local trauma to the vasculature), a small stab wound was made through the sclera just dorsal to the optic nerve, severing only the axons in the superior quadrant of the retina (n = 12 animals). When whole

CAP-43 6 17

Figure

in the CNS

2. GAP43

lmmunofluorescence

in \nWhole-Mount

Retinas

as an Assay

for Chang

es in GAP-43

row) rats. Whole (top row), l&day-old (m .iddle row), or adult (bottom .Anti-GAP-43 binding to whole mounts of ret :inas from l-day-old followed by secondary or preimm une IgG (third column), mounts were incubated with anti-GAP-43 (3.t j pglml; first and second columns) Only retinas of l-day-old rats (a (first column) or fluorescein (second and th ird columns). antibodies conjugated to either rhodamine and b) were stained intensely. Bar, 200 urn.

NeLlRXl 638

WHOLE

MOUNTS

WHOLE

MOUNTS

4308

8 Q

b , 0

I

I

IO

I,

20 DAYS

Figure 3. Summary Axotomy-Induced

AFTER

INTRAORBITAL

AFTER

AXOTOMY

INTRACRANIAL

conclude that the increase in antibody labeling is a consequence of axotomy. In addition to the retinal axons, an increased number of cell bodies were reactive with anti-GAP-43 between 6 and 20 days after axotomy;asinthecaseoftheaxons,thefluorescent labeling was punctate (Figures 5b-5d). Theseexperimentsshowthatthereactivityof retinal whole mounts with anti-GAP-43 antibodies increases after axotomy close to the eye, but not if the position of the axotomy is more than 6 mm from the eye.

AXOTOMY

03 I

I

30

I

AFTER

I1

40

1

50

I

I1

60

I

70

AXOTOMY

of Time Course and Position Anti-GAP-43 Labeling of Adult

Depenence Rat Retinas

of

Open circles (intraorbital axotomy 1-3 mm from the eye) and diamonds(intracranialaxotomy6-IOmmfrom theeye)represent retinas in which labeling with anti-GAP-43 was indistinguishable from controls funoperated retinas) (Figure 4b). Closed symbols represent retinas that were more highly labeled with anti-GAP-43 than retinas from unoperated control animals (e.g., see Figure 4a). Enhanced anti-GAP-43 labeling was apparent only between 6 and 26 days after axotomy (hatched region) and only if the axotomy was within 3 mm of the eye.

mounts of these retinas were labeled with anti-GAP-43 13 days later, the fluorescent labeling had increased only in the axons peripheral to the lesion (i.e., between the lesion and the cell body) in the superior quadrant of the retina; no increase was detected in axons that were not interrupted by the lesion. We

Figure

4. GAP-43

lmmunofluorescence

in Axotomized

and

Control

Anti-GAP43 (5.6 pg/ml) binding to whole mounts of adult rat retina a control retina (unoperated side; b). OD, optic disk. Bar, 200 pm.

Radiolabeling of Axonally after Axotomy

Transported

GAP-43

To investigate whether the amount of newly synthesized GAP-43 in retinal ganglion cells changes after axotomy, we injected [?S]methionine into the vitreous of adult rats and 3 hr later extracted the proteins from the optic nerve stump and separated them by two-dimensional gel electrophoresis (IEF-SDS-PAGE). (It was necessary to analyze GAP-43 in the optic nerve stump rather than in the whole retina to facilitate the identification of GAP-43 and to insure that its source was the retinal nal neurons beled, axonally by exposing to X-ray film; fluorographs son with the visualized by

ganglion cells, which are the only retithat project to the optic nerve.) The latransported proteins were detected the gels (impregnated with scintillators) the position of GAP-43 on the resulting (Figure 6d) was determined by compariposition of purified GAP-43 on the gel, Coomassie blue staining (Figure 6b). The

Retinas 12 days after

axotomy

within

3 mm of the eye (a) compared

with

r;AP-43 r,39

in the CNS

Figure

5. Whole

Mounted

At low magnification fluorescent labeling labeling of individual

Adult

Rat Retina

Labeled

with

Anti-CAP-43

Antibody

12 Days

after

Axotomy

Close

to the

Eye

([a], bar, 200 pm) labeled axon bundles and somata are evident. At higher magnification ([b-d]; bar, often appeared granular and filled the cytoplasm and proximal dendrite (b), but not the nuclei (arrows axons was often discontinuous, producing a beaded appearance (d, arrowheads].

5 pm) the in c]. The

Neuron 640

Figure 6. Identification of GAP-43 on Fluorographs of Two-Dimensional IEF-SDSPolyacrylamide Gels (a) and (b) show Coomassie blue-stained proteins from normal rat optic nerve with (b) or without (a) the addition of purified GAP-43 (2.5 pg) to mark the position of GAP-43 (43) relative to tubulin (a and 0) and actin (a in figures). (d) is a fluorograph showing that a protein with the electrophoretie mobility of GAP-43 as well as Superprotein (SP), a major axonally transported protein,

are labeled

nine was injected

3 hr after

P%]methio-

into theeye

in the stump

of an optic nerve that was axotomized 12 days previously. (c) is a fluorograph showing that, unlike tubulin and actin, neither CAP-43 nor SP was labeled 3 hr after [35S]methionine was injected into the optic nerve of a rat.

b

amount of radioactivity associated with GAP-43 was estimated by scanning the appropriate spot with a laser densitometer, as described in Experimental Procedures. The following observations indicated that the amount of newly synthesized GAP-43 increased after axotomy close to the eye. First, labeled GAP-43 could be detected on fluorographs after exposure to less total radioactivity (see Experimental Procedures) in samples from axotomized rats than in samples from control rats, indicating that GAP-43 represented a larger fraction of the total radioactivity after axotomy. For example, GAP-43 was detected in an axotomized optic nerve after the fluorograph was exposed to 1 x IO9 radioactive disintegrations (Figure 7a), but was not detected in the sample from the intact optic nerve after 1.7 x IO9 disintegrations (Figure 7~). However, because only a short stump of optic nerve close to the site of injection was available for analysis, a significant and variable portion of the total radioactivity in the sample was a consequence of local incorporation of isotope stump.

into proteins Therefore, we

by nonneuronal compared the

cells amount

in the of radio-

activity associated with GAP-43 with the amount ciated with another major axonally transported tein, designated Superprotein et al., 1974). Neither Superprotein

beled detectably

(protein nor

when [35S]methionine

rectly into the optic (Figure 6c), indicating associated with either

assopro-

#20 in Willard GAP-43 are la-

is injected

di-

nerve, instead of the vitreous that the presence of label of these proteins in the optic

nerve is a consequence of their transport in retinal ganglion cell axons. An increase in the ratio of GAP-43 to Superprotein following axotomy is evident from

inspection of Figure 7, in which afluorograph exposed for long enough to detect GAP-43 from an optic nerve with axotomized 12 days previously (a) is compared increasingly longer exposures of fluorographs from normal intact controls (b-d). It is apparent that the spot corresponding to Superprotein from the control nerve is darker on a fluorograph exposed for insufficient time to detect GAP-43 (Figure 7c) than on a fluorograph from the axotomized nerve that reveals GAP-43 (Figure 7a). (GAP-43 can be detected in the control nerve after longer exposure [Figure 7d].) Measurements (see Experimental Procedures) of the relative densities of the fluorographic spots at different times after axotomy (Table 1) show that, one day after axotomy 2-3 mm from the eye, the ratio of GAP-43 to Superprotein radioactivity was the same as in control rats after

that

were

not

axotomized.

However,

at 12 days

axotomy, the ratio of GAP-43 to Superprotein was 22-fold higherthan in the unaxotomized controls. By 21 days, the ratio was only4.4 times higher. On the other

hand,

when

ally IO mm behind later

the

ratio

the

of GAP-43

times that of the labeling of newly of Superprotein

that this increase to the eye.

optic

nerve

was

the eye (Figure

cut

intracrani-

7E; n = I), 12 days

to Superprotein

controls. These results synthesized GAP-43

was show relative

only

1.4

that the to that

increases after axotomy and suggest occurs only when the injury is close

C4P-43 611

in the CNS

Figure 7. Axonally Axotomized and

Transported Intact Optic

GAP-43 Nerves

in

Fluorographs of two-dimensional gels showing an increased ratio of labeled GAP-43 (43) relative to Superprotein (SP), another rapidly transported protein, 12 days after axotomy (a) compared with unaxotomized controls(b-d). lncreasingfluorographicexposures of the unaxotomized controls shown in (b)-(d) illustrate that the spot corresponding to Superprotein is darker on a fluorograph of a control sample that does not reveal GAP43(c) than on a fluorograph of a sample from an axotomized nerve that does reveal CAP-43 (a). (E) shows the proteins transported into the optic nerve 12 days after intracranial axotomy, 10 mm from the eye; the ratio, GAP-43: Superprotein, is similar to that observed in unaxotomized controls (see text). Tubulin and actin (arrows in [a]), presumably labeled by synthesis in the optic nerve, were not ap parent in the unaxotomized controls because the 3 mm of optic nerve closest to the eye was not analyzed. In all cases, p5lmethionine was injected into the vitre ous 3 hr before the animals were killed. Fluorographs were exposed to the following total disintegrations: (see Experimental Procedures) (a), 1.0 x 109; (b), 1.2 x 108; (c), 1.7 x 109; (d), 4.3 x 109; (e) 8.9 x W.

E

Discussion iable 1. Change in Labeling after Axotomy of the Optic

of Axonally Nerve

Transported

GAP-43

Days after hxotomy

Position of Axotomy”

Ratio (x IOY GAP-43: Superprotein

Ratio’ Axotomy: Control

N (Animals)

( 1 12 20, 21 12

Proximal Proximal Proximal Proximal Distal

4.6 * 0.76 4.3 102 f 42 20.4 f 1.8 6.4

1.0 .09 22.0 f 5.4 4.4 * 0.34 1.4

4 1 3 3 1

a Proximal eye. Distal t Determined scribed in c Previous

axotomy was intraorbital, less than 3 mm from the axotomy was intracranial, 10 mm from the eye. by densitometric scanning of fluorograms, as deExperimental Procedures. column has been normalized to control (0 days).

The experiments reported here showed that the reactivityof whole mounts of rat retina to anti-GAP-43 antibodies was elevated between 6 and 20 days after axotomy of the optic nerve, but only if the position of the axotomy was close to the eye; reactivity did not increase after intracranial axotomy 6 mm or farther from the eye. The relative labeling of axonally transported GAP-43 with [35S]methionine also increased, with a similar time course and similar dependence upon position of the axotomy. Several influences could potentially contribute to these responses. For example, the change in ratio (approximately 20-fold) of [35S]methionine-labeled GAP-43 to another rapidly

transported protein, Superprotein, could reflect either an increase in newly synthesized GAP-43 or a decrease in newly synthesized Superprotein. However, the radiolabeling of Superprotein has been observed to be relatively insensitive to axotomy; it has been reported to increase approximately 3-fold after axotomy of toad retinal ganglion cells (Skene and Willard, 1981a), to be unchanged after axotomy of rabbit retinal ganglion cells near the optic chiasm (Skene and Willard, 1981b) or rat corticospinal tract axons at the level of the pyramids (Reh et al., 1987), and to decline by a factor of 2 after axotomy of rat peripheral nerve (Reh et al., 1987). (In the current experiments we observed no evidence for a change in the rate of synthesis of Superprotein, but the efficiency of labeling was not sufficiently constant to address this question rigorously.) It is therefore likely that the change in ratio is primarily a consequence of an increase in recently synthesized GAP-43. It is unlikely that this increase is solely a consequence of events that occur when GAP-43 accumulates at the site of axotomy, because no change in this ratio was observed 1 day after axotomy close to the eye or 12 days after intracranial axotomy. Nor is it probable that this increase is a consequence of a decrease in the rate of degradation of GAP-43; because GAP-43 is normally degraded slowly (a half-life of approximately 1 week has been estimated Uacobson et al., 1986]), a reduction in this rate would not produce an appreciable change in the amount of radiolabeled GAP-43 measured 3 hr after its synthesis. These considerations suggest that the increase in newly synthesized GAP-43 relative to Superprotein reflects an increase in either the rate of synthesis or axonal transport of GAP-43. The difference in intensity of anti-GAP-43 staining of retinal whole mounts in proximally axotomized rats compared with controls (Figure 4) resembled the difference between l-day-old rats and adults (Figure 2). During this period (1 day postnatal to adulthood), the amount of recently synthesized GAP-43 transported into the optic nerve decreases at least 20-fold in rabbits (Skene and Willard, 1981b) and similarly in rats (Freeman et al., 1986). The increased staining after axotomy may reflect changes in the concentration of GAP-43 of this magnitude. However, because the assay was not sufficiently sensitive to detect GAP-43 in normal adult retina, we do not know its threshold. Furthermore, in neither the case of development or axotomy can we exclude the possibility that factors other than the concentration of GAP-43, such as changes in antigenicitydue to posttranslational modification, may contribute to the observed changes in immunofluorescence of the retinal whole mounts. It should also be noted that changes other than increased synthesis could potentially contribute to an increased concentration of GAP-43 in the retina after axotomy close to the eye: for example, the accumulation in the retina of GAP-43 that would normally be transported to the distal portion of the axons. However, this alternative mechanism might not ade-

quately account for the delay of approximately 6 days between axotomy and increased immunoreactivity of the retina. If this delay represented the time required to saturate the extraretinal axon stump with GAP-43 then it might be anticipated that the intraretinal axons, someofwhichare3-4mm long,would becomeimmunoreactive in a distal to proximal sequence over a period of several days. On the contrary, all of the retinal axons appeared uniformly reactive or unreactive along their entire length, suggesting that additional factors (e.g., increased synthesis of GAP-43) are important in generating the increased immunostaining after proximal axotomy. Thus, although other possibilities have not been excluded, a parsimonious and plausible explanation for the delayed increase in the immunoreactivity of the retina and the GAP-43:Superprotein ratio is that both reflect at least in part an increase in the rate of synthesis of GAP-43, beginning between 1 and 6 days after proximal axotomy and reaching a level of approximately 20-fold by 12 days after axotomy. The subsequent decline in both immunoreactivity and the GAP-43:Superprotein ratio may reflect in part changes related to the eventual demise of the axotomized neuron. However, we have observed that the immunoreactivity of the retina changes with a similar time course when the proximally injured axons are permitted to regenerate into a peripheral nerve graft (Lozano et al., 1987, Sot. Neurosci., abstract), a process that prolongs the lifetime of the regenerating neurons (Villegas-Perez et al., 1988). Changes in the GAP43:Superprotein ratio similar to those reported here also occur when regeneration is permitted; after the decline, its value may remain several fold higher than in the unaxotomized control (Doster et al., 1988, Sot. Neurosci., abstract; Wouters and Norden, 1990, Sot. Neurosci., abstract). Thus, the decline in these measures may also occur in viable neurons that are regenerating axons. It has been suggested that the induction of GAP-43 may be one aspect of establishing a metabolic state in which a neuron has the potential to grow an axon efficiently (e.g., Skene and Willard, 1981a, 1981 b; Willard et al., 1985). Although the current experiments demonstrate conclusively that GAP-43 can be induced in neurons in situations in which they do not regenerate their axons, the position dependence of this response suggests a correlation between the induction of GAP-43 and the potential for axon growth. Although mammalian neurons do not normally regenerate injured axons in the CNS, they do so when provided with a peripheral nerve graft (for review, see Aguayo, 1985; Aguayo et al., 1990). However, this regeneration only occurs when the neurons are injured close to their cell bodies (David and Aguayo, 1981; Benfeyand Aguayo, 1982; Richardson et al., 1984). In particular, axons of adult rat retinal ganglion cells will regenerate for a distance of several centimeters into a segment of peripheral nerve grafted into the retina or optic disk (So and Aguayo, 1985; Vidal-Sanz et al., 1987), but

G.IP-43 643

in the CNS

will not regenerate into a graft placed in the optic nerve approximately 8 mm from the eye (Richardson et al., 1982). This correlation between the position dependence of the induction of GAP-43 and regeneration into a peripheral nerve graft is consistent with the hypothesis that the induction of GAP-43 is a marker for metabolic changes that enhance the potential of the CNS neuron for axon growth; however, this potential is realized onlywhen an appropriateenvironment, such as a peripheral nerve graft, is provided. The dependence of GAP-43 induction upon the position of axotomy reported here may explain why GAP-43 induction was not observed previously after the adult mammalian optic nerve was injured near the optic chiasm (Skene and Willard, 1981b) or after the corticospinal tract of adult hamsters (Kalil and Skene, 1986) or rats (Reh et al., 1987) was lesioned in the brain stem at the level of the pyramids, but GAP-43 induction was observed 12 days after axotomy of the rat optic nerve (Freeman et al., 1986). In addition, increased GAP-43 immunoreactivity has been observed near infarcts (Ng et al., 1988) and lesions of the brain (Foerster, 1988, Sot. Neurosci., abstract); this may reflect increased GAP-43 synthesis in nearby cells whose axons have been injured. The induction of GAP-43 by axon injury and its position dependence has implications for the question of how GAP-43 expression is regulated. First, it shows that interruption of the axons of CNS neurons is sufficient to induce GAP-43; for example, inducing factors supplied by peripheral nerve grafts are not required. Second, the inverse correlation between the distance of axotomy from the eye and its ability to induce GAP43 suggestsasimple mechanism that might contribute to the regulation of GAP-43 in this system. According to this hypothetical mechanism, GAP-43 synthesis would normally be repressed in the cell bodies by elements derived from nonneuronal cells of the optic perve; these repressive elements (or secondary signals generated by them) would require an intact length of axon to serve as a conduit between their source and the retinal ganglion cell body. Thus after axotomy, the level of repressor in the cell body would cepend upon the length of intact axon remaining accessible to nonneuronal components of the optic rlerve. Injuries that interrupted the axons close to tne eye might reduce the level of such repressive c-lements reaching the cell body sufficiently to derepress GAP-43 synthesis. This hypothetical mechanism would predict that the components of the optic nerve, come of which have been observed to inhibit the adhesiveness and extension of growth cones (Caroni ;‘nd Schwab, 1988; Schwab, 1990; Carbonetto et al., :987), include elements that lead to repression of GAP-43 expression. It also predicts that, although GAP-43 repression would depend upon the length of the intact axon in the optic nerve, it would not necessarily depend upon the length of axon within the retina, where the population of nonneuronal cells is different than in the optic nerve. The length of the

intraretinal segments of these axons varies by 3-4 mm according to whether the retinal ganglion cell is located in the periphery of the retina or close to the optic disc. In fact, we observed that axotomies less than 3 mm from the eye always lead to reactivity of retinal ganglion cell axons in the entire retina, even though the remaining axons of the peripheral cell bodies may be 6-7 mm long, whereas axotomies 6 mm from the eye never generated antibody staining, even in the central portion of the retina where the remaining axons are shortest. Although this hypothetical mechanism could explain the position dependence of GAP-43 induction by axotomy observed here, certain caveats should be noted: First, alternative mechanisms, such as the generation by axotomy of positive influences that are only effective in inducing GAP-43 over short distances, cannot be ruled out. Second, the time during which a long length of intact axon is exposed to nonneuronal cells(and hencethe hypothetical repressiveelements) of the optic nerve after distal axotomy is limited, because axons severed far from their soma tend to undergo extensive retrograde degeneration (Richardson et al., 1982). Third, certain neurons within the brain of adult mammals express high basal levels of GAP-43 relative to the retinal ganglion cells (Benowitz et al., 1988), suggesting that repression of GAP-43 by this mechanism would be differentially effective within the CNS. Fourth, in peripheral nerve, GAP-43 induction and axon growth do not appear to depend upon the position of axotomy, suggesting that they are not effectively repressed by the population of nonneuronal cells in the peripheral nerve; however, this population differs from that of the CNS, notably in the presence of Schwann cells and absence of astrocytes and oligondendrocytes. Fifth, GAP-43 (and axon growth) can be induced in sensory neurons of the dorsal root ganglion after axotomy of one branch of their axons in the peripheral nerve, even though the intact central branch maintains an association with nonneuronal cells of the CNS. On the other hand, the central branches of the axons of lumbar dorsal root ganglia neurons regenerate large numbers of their spinal cord projections into nerve grafts inserted into the cervical spinal cord only when the peripheral branches of these neurons are also severed (Richardson and Issa, 1984), a procedure that enhances the expression of GAP-43 (Schreyer and Skene, 1988, Sot. Neurosi., abstract; Woolf et al., 1990). These caveats suggest that, if GAP-43 can in fact be repressed by nonneuronal elements of the CNS, this is not the only mechanism for regulating GAP-43 synthesis and that, under certain conditions, other mechanisms may override this represssion. Experimental

Procedures

Purification of GAP-43 CAP-43 was purified from l-day-old rat brains by a procedure involving differential extraction with SDS and precipitation with chloroform-methanol (described in Changellian et al., 1990) fol-

NellrOn 644

lowed by either preparativetwo-dimensional gel electrophoresis (described below) or reversed-phase chromatography on a C-4 high pressure liquid chromatography column (described in Changellian et al., 1990). Preparative two-dimensional gel electrophore sis was performed as follows. After precipitation with chloroform and methanol, the pellet (from eight brains) was sonicated, then incubated at 100°C for 10 min in solution I (1% [w/v] SDS, 2.5 mM dithiothreitol; 300 ul per pellet). An equal volume of solution II (10% [v/v] Nonidet P-40, 25 mM dithiothreitol) and 0.468 g of urea per 600 ul were added, and the solution was clarified by centrifugation at 100,000 x g in a Beckman Airfuge for 10 min and subjected to electrofocusing on polyacrylamide gels (O’Farrell, 1975) 11.5 cm high x 1.5 cm thick containing 16.5 g of urea, 12 ml of HIO, 1.8 ml of ampholytes (pH 3.5-10; LKB Instruments, Inc.), 4 ml of a solution containing 30% acrylamide and 1.6% bisacrylamide, 600 ul of Nonidet P-40,60 ~1 of TEMED, and 90 ul of 7% ammonium persulfate (7% [w/v]). Prior to application of the sample, thegel was overlaid with solution II and prefocused for 15 min at 200 V, 15 min at 300 V, and 30 min at 400 V. The upper solutions were removed; the sample was placed in a large single well and overlaid with solution II and focused for 20 hr at 440 V. The gels were then cut into 1 cm horizontal strips and frozen in equilibrium buffer (100 mM Tris [pH 8.01, 2% SDS, 2 mM dithiothreitol, 10% glycerol, bromophenol blue). A small piece of gel from each strip was run on an analytical SDS-polyacrylamide gel, which was stained with silver by the method of Merril et al. (1981) to locate GAP-43. The strips of focusing gel that contained GAP43 were then layered over SDS-polyacryamide gels (7% acrylamide), which were run for 810 V-hr using the buffer system of Laemmli (1970). Proteins on these gels were visualized by incubation in 4 M sodium acetate for 15 min, and GAP43was identified by comparison with the silver-stained gels. The band containing GAP-43 was cut out and eluted in an electroeluter at 3 W for 24 hr. GAP-43 was then precipitated with chloroform-methanol (Wessel and Flugge, 1984). This procedure, which was used initially, proved more cumbersome and less reliable than the procedure using reversed-phase chromatography (described in Changellian et al., 1990), which was used subsequently. The identity and purity of the protein was confirmed by gel electrophoresis and (in the case of GAP-43 purified by the reverse-phase method) analysis of the amino acid composition using the method of Bidlingmeyer et al. (1984). In addition, the average phosphoserine content of the preparation was estimated to beapproximately mol per mol of GAP-43; phosphoserine was identified by chromatography of an acid hydrolysate of 5 ug of GAP-43 (purified by the reverse-phase chromatography method) on an Altex DEAE 5-PW column (7.5 x 7.5 mm), using a linear gradient from 100% buffer A (0.01 M NaBOs [pH 11.21) to 100% buffer B (0.05 M NaBO,, 0.7 M NaCl [pH ll.O]). Antibodies to CAP-43 Approximately 100 ug of GAP43 (purified by preparative twodimensional gels) was emulsified in 1 ml of Freund’s complete adjuvantand injected intradermallyat multiplesiteson the backs of rabbits. Boosting injections consisting of approximately 100 pg of GAP-43 (purified by reversed-phase chromatography) either emulsified in RIBI MPL + TDM adjuvant (in two rabbits) or in Freund’s incomplete adjuvant (in two rabbits) were given at approximately 1 month intervals. Serum was obtained 10 days after each injection. The rabbits that received the RIBI adjuvant developed IO-fold higher titers of antibody. An IgG-enriched fraction was precipitated from the serum with 50% ammonium sulfate, dialyzed against PBS (0.15 M sodium chloride, 0.01 M sodium phosphate [pH 7.4]), and purified by affinity chromatography on a GAP43-Sepharose 48 column. The affinity column was prepared by incubating 800 ug of purified GAP-43 (purified by reverse-phase chromatography) with 0.3gof cyanogen bromide-activated Sepharose4B beads(Sigma) in coupling buffer (0.1 M sodium bicarbonate, 0.5 M sodium chloride [pH 8.31) overnight at 4” C. After application of the IgGenriched serum fractions, the column was washed with 10 ml of PBS, and the antibodies were eluted with 10 ml of 0.2 M glycine

(pH 2.4). Fractions (1 ml) were collected in tubes containing 55 ~1 of 2 M Tris (pH 8) to neutralize the glycine. The absorption of 280 nm was monitored to identify the samples containing protein. Preimmune antibody was prepared by precipitation with ammonium sulfate (50% [w/v]) of serum drawn from each rabbit prior to the first immunization. The ammonium sulfate precipitate was dialyzed against PBS and stored at -7OOC. All immunohistochemistry described here was performed with affinitypurified antibody from a single preparation, and preimmune antibody from the same rabbit was used for all preimmune controls. The antibody was characterized by Western blotting. After separation by SDS-PAGE, proteins were transferred to nitrocellulosepaperthatwas incubatedwith antiGAP43antibody(l ml). Antibody binding was detected by thevectastain ABC procedure (Vector Laboratories, Burlingame, CA). Incubation with a secondary antibody conjugated to biotin was followed by incubation with avidin conjugated to horseradish peroxidase, whose reaction product with diaminobenzidine was visualized. AntiCAP-43 used in these experiments reacted with a single polypeptide band on Western blots of a homogenate of neonatal rat brain fractionated on one-dimensional SDS-polyacrylamide gels (Figure 7), but only when the gels were first incubated for 30 min in a solution of 40% methanol and 10% acetic acid and then soaked in 1% SDS in transfer buffer (20 mM Tris, 150 mM glycine, 20% methanol) for 30 min prior to transfer for 30 min at 120 V in transfer buffer, using a Bio-Rad transfer apparatus. Theenhancement of transfer of CAP-43 by this procedure has been described previously (Meiri et al., 1986). The antibody did not react with any of the proteins that were transferred to nitrocellulose from gels that were not treated in this way. On two-dimensional IEF-SDS-PAGE Western blots, the antrbody reacted with several spots (or a streak, depending upon the amount of protein in the sample) with both acidic pl and electrophoretic mobility similar to purified GAP-43. These reactive spots comigrated with purified GAP43 on SDS-polyacrylamide gels containing either 7% or 10% acrylamide, indicating that the reactive material had the same unusual dependence upon acrylamide concentration as did authentic GAP-43, whose electrophoretic mobility corresponds to an apparent molecular weight of 54 kd and 48 kd on 7% and 10% gels, respectively. (This unusual characteristic of GAP-43 has been attributed to its binding less than the average amount of SDS per unit of mass Uacobson et al., 1986; Benowitz et al., 1987.) Antibody Binding to Whole Mounts of Retina Rats were anesthetized with 3.5% chloral hydrate and perfused through the heart (after cross-clamping the thoracic aorta) with 200 ml of PBS, followed by 300 ml of 4% paraformaldehyde in PBS. The retinas were removed as whole mounts, postfixed on filter paper in 4% paraformaldehyde for 45 min, then rinsed by floating in PBS for 2 hr. The retinas were incubated overnight by floating them in affinity-purified rabbit anti-CAP-13 [email protected] or 4.6 pg/ml), 10% goat serum, and 2% Triton X-100 in PBS, then washed with PBS, incubated with fluorescein isothiocyanate goat anti-rabbit IgC for 2 hr, washed again with PBS, and mounted in Citifluor. Some retinas were double labeled with mouse monoclonal antibodies to neurofilament-H (RT97 at I:1000 dilution), following the same protocol, prior to mounting. Controls were stained under identical conditions using preimmune antibody (at the same or higher concentrations of antibody: 2.8-560 ug/ml) from the same rabbit. Shorter incubations in primary antibody resulted in no staining, as did lower concentrations of Triton X-100. Higher concentrations of Triton X-100 increased the nonspecific staining, without affectingthespecific staining. Freezing and thawing of the whole mounts to expose intracellular antigens did not affect the staining. Surgical Procedures Adult female Sprague-Dawley rats weighing 200-300 g were anesthetized by intraperitoneal injection of 3.5% chloral hydrate (1 ml per 100 g) for all surgical procedures, including intravitreal injections. The left eyewas operated upon, and the right eye was used as an unoperated control. Before and after surgery, the

:,P-43

in the CNS

eyes were dilated with a 1% ophthalmic solution of Cyclogyl, and a drop of saline was placed on the eyes to facilitate examination of the retina under the dissecting microscope. If evidence cf infarction or signs of vascular compromise were present, the animals were not used. Animals that developed cataracts after surgery had thin, fibrous, acellular retinas that were scarred to the sclera; therefore, cataracts were considered indicative of inf,lrction. After surgery, the eyes were protected with Neosporin ointment. lntraorbital axotomy was performed as follows: The scalp was incised at the midline and retracted on both sides, and the periosteum was incised along the rim of the orbit and retracted laterally to expose the orbital contents. The supraorbital nerve and extraocular muscles were cut to expose the optic nerve. The sheath of the optic nerve was opened longitudinally along its dorsal aspect, and a nerve hook was placed around the nerve within the sheath. The optic nerve was cut where it rested on the hook, and care was taken not to disturb the central artery, which courses along the ventral aspect of the sheath. The periosteum was closed with 6-O suture, and the scalp was closed with Michel clips. For intraretinal axotomy, the orbit was exposed as above, and the top of a 19 gauge needle was passed through the sclera just dorsal to the optic nerve, cutting the axons from the superior quadrant of the retina. In thecase of intraretinal axotomy, direct injury to the lens was unavoidable, and cataracts that developed in these animals were not considered indicative of vascular damage. For intracranial axotomy, the scalp was incised as above, and ii frontal craniectomy and partial frontal lobectomy were performed to expose the optic nerve, which was cut along the floor of the skull. The defect was filled with gelfoam, and the scalp was closed with Michel clips. Labeling of Axonally Transported Proteins [‘5S]methionine (0.3 mCi, >lOOO Ci/mmol in a volume of 6 pl of water) was injected into thevitreous of rats to label newly synthe !.ized proteins in retinal cells. Three hours later, the animals were killed by intraperitoneal injection of a lethal dose of chloryl Ilydrate, and the optic nerves were removed, frozen on dry ice, and stored at -70°C. The tissue was homogenized in 1 ml of ice-cold H-buffer (5 mM IiDTA, 2.5 mM phenylmethylsulphonyl fluoride, 10 mM Tris [pH ‘.4]) per sample and centrifuged at 100,000 x g for 1 hr. The .nembrane pellets were sonicated for 5 s in solubilizing buffer (50 ~1 per optic nerve) and then incubated for 10 min at 100°C. 4n equal volume of solubilizing buffer II and 0.078 g of urea per 100 ~1 were added. Samples were mixed on a Vortex mixer and rhen clarified by centrifugation for 30 s in a Beckman Airfuge. An lliquot (50 ~1) of sample was added to 1 ml of 10% trichloroacetic acid and vacuum-filtered through nitrocellulose membranes (0.45 pm pore size). The filters were washed three times with 1 ml of 5% trichloroacetic acid and air dried, and radioactivity was determined by liquid scintillation counting. The samples were electrophoresed on two-dimensional gels. The first dimension was isoelectric focusing for 7000-8000 V-hr ~:O’Farrell, 1975), using as ampholytes a mixture of 50% pH 3.510 and 50% pH 5-7 ampholytes from LKB Instruments, inc., followed by SDS-PAGE on 10% acrylamide slab gels, using the buffer system of Laemmli (1970).Thegelswere impregnated with ErQHance (New England Nuclear Corporation), dried at reduced pressure on filter paper, and exposed -70°C to Kodak XAR-film, which had been pre-exposed to an optical density of 0.15 relative to unexposed film (Laskey and Mills, 1975). To compare different fluorographs, we derived the following expression for the total number of disintegrations (D) that occurred during the fluorographic exposure: D = (1.83 x 10S)([DPM], - [DPM],e*.m7881), in which [DPM], is the radioactivity (DPM) in the sample applied to the gel at the beginning of the exposure, and t is the duration of the exposure in days. Approximately 2 x IO9 and 2 x IO’O disintegrations were required to visualize CAP-43 from unoperated optic nerve and retina, respectively. To measure the amount of radioactivity associated with GAP-

43 and the rapidly transported protein Superprotein, the appropriate spots on the fluorographs were scanned with a laser densitometer (LKB 2222-010 Ultrascan XL) programmed to average the data over 80 Km in the x direction and 240 pm in they direction, and the total optical density of each spot was calculated using LKB software (Gel Scan XL Laser Densitometer Program Version 1.21) run on a Dell Systems 200 computer. The average optical density of the perimeter of the region that was scanned served as background. Only spots whose optical densities were within the linear range of the densitometer (appropriately O-4 absorbance units) were included. To verify empirically that the absorbance determined by this means was a suitable measure of the radioactivity, different autoradiographic exposures of the same gel were analyzed, showing that the calculated optical densities of the spots were proportional to the total disintegrations (D) during the exposure. Acknowledgments We thank the following people: Karina Meiri, who participated in preliminary phases of this work; M. VidaCSanz, Susan Schuh, Susan Spencer, Arleen Loewy, Lora Beasley, Margaret David, Susan Shinn, Wendy Wilcox, and Charles Essagian for advice, discussions, and technical input; Ursula Drager for advice on mount procedures; and Rose Hays and Janis Branneky for typing the manuscript. This work was supported by grants from NIH (EY06082), the McKnight Foundation, the Medical Research Council of Canada, and the Multiple Sclerosis Society of Canada. A. 1. A. is a member of the Canadian network for the study of neural regeneration and functional recovery. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement”in accordance with 18 USC Section 1734 solely to indicate this fact. Received

October

15, 1990; revised

December

26, 1990.

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