Molecular Bram Research, 14 (1992) 147-153
147
Elsewer BRESM 80128
Synthesis and transport of GAP-43 in entorhinal cortex neurons and perforant pathway during lesion-induced sprouting and reactive synaptogenesis Li-Hsien Lin, Susan Bock, Kenneth Carpenter, Michael Rose and Jeanette J. Norden Department of Cell Biology, Vanderbdt Universtty Medwal School, Nashvdle, TN 37232 (USA)
(Accepted 11 February 1992) Key words" Growth-associated protein; Synaptlc plasticity; Hlppocampus; Axonal transport; Synaptosomal-assocmtedprotein
Metabohc labehng and quantitative 2D gel autoradlography were used to assess changes m the synthes~s and transport of GAP-43 m entorhmal cortex (EC) neurons and perforant pathway during lesion-reduced sprouting and reactive synaptogenesls. In normal adult rats, there Is a high constltutwe level of GAP-43 synthesis and transport m EC neurons projecting to the hlppocampus Following umlateral EC lesions, there is a 2-fold (100%) increase m the transport of newly synthesized GAP-43 to the contralateral or 'sprouting" hippocampus. The txmmg of this upregulatlon (between 6 and 15 days) suggests that changes in GAP-43 expression occur m response to the growth of presynaptlc terminals during sprouting Growth-associated protein-43 (GAP-43; B-50:7; Fl46; pp4628; neuromodulin 5°) is a fast-axonally transported phosphoprotein postulated to play a role in axon growth and synaptlc plasticity (reviewed in refs. 3,12,23,32,33, 41,45). During development 12n8'43 and regeneration 1'42, axon growth is correlated with a significant upregulation of GAP-43 synthesis and transport. Consistent with these findings, GAP-43 is localized to fiber pathways in the developing brain 26 and concentrated in growth cones 9'44. In adult animals, GAP-43 is concentrated in axon terminals 4'26, particularly within so-called 'plastic' areas such as the neocortex, cerebellum and hippocampus 4'z6, where it is believed to play a role in synaptic plasticity. For example, in the hippocampus, the posttranslational modification of GAP-43 has been linked to changes in the efficacy of synapses. In long-term potentiation (LTP), there is a significant increase in the in vitro phosphorylation of GAP-43 (F146) specifically linked to the maintenance of LTP z4'25'38 Our lab has been interested in examining the role GAP-43 may play in another form of synaptic plasticity in the adult brain. This type of plasticity, which is termed lesioned-induced sprouting and reactive synaptogenesis, involves the terminal growth of normal axons in response to the removal of other afferents and is particularly robust in the hippocampus (reviewed in refs. 7,8). For example, in adult rats there is a major projection of the entorhinal cortex (EC) to the outer 2/3-3/4 of the
molecular layer of the ipsilateral dentate gyrus and a very minor projection to the contralateral side 47. Following unilateral removal of the EC, the normally sparse contralateral EC projection, commissural/associational fibers, and other normal afferents, 'sprout' axon terminals and form new synapses in the denervated portion of the dentate molecular layer 47. Although the 'sprouting' paradigm is widely used to study synaptic plasticity, only one study has addressed the issue of changes in presynaptic protein synthesis and transport during this process. Using light-microscopic autoradiography to visualize all 3H-labeled proteins transported in commissural/ associational afferents following EC lesions, Goldowitz and Cotman 14 found no change in the labeling pattern of fast-axonally transported proteins during sprouting. While the overall labeling pattern of proteins might not change during sprouting and reactive synaptogenesis, the expression of individual proteins may be regulated during these processes. Here we have re-examined this question by quantitating changes in the expression of a specific protein which is postulated to play a role in both axon growth and synaptic plasticity. Using 2D gel autoradiography we have quantitated changes over a timecourse in the synthesis and transport of GAP-43 in EC neurons and perforant pathway during terminal sprouting (4-6 days) and reactive synaptogenesis (15 days). Sixteen adult male Sprague-Dawley rats (250-330 g) were anesthetized with ketamine (87 mg/kg) and xyla-
Correspondence J J Norden, Department of Cell Biology, Vanderbllt Umversity Me&cal School. Nashvdle. TN 37232-2175, USA Fax: (1)
(615) 343-4539
148 zme (13 mg/kg). In normal (unlesioned) ammals, the E C was exposed on one u d e and injecnons of 0,5 #1 of [35S]methionine were m a d e into the medial E C 44 with a Hamilton microliter syringe. Injections were made over a 2-rain period and the syringe was left in place for an additional 10 min Animals were sacrificed by drug overdose (Nembutal; 50 mg/kg) at 2, 3, 6 or 12 h following the last injection. Both dorsal hlppocampi were removed, and high-speed m e m b r a n e fractions were p r e p a r e d and subjected to 2 D - P A G E autoradiography as described previously 12. F r o m these data in normal animals, we determined that 6 h was the optimal time for labehng of proteins to the contralateral hippocampus by fast-axonal transport. In experimental ammals, the right EC was removed by aspiratmn, and the animals allowed to survive for 4 - 6 or 15 days. A t the appropriate time, the animal was re-anesthetized, [35S]methionlne rejections were made into the remaining intact EC, and the dorsal hippocampi and dentate gyrl were r e m o v e d 6 h later and subjected to 2 D - P A G E autoradiography. Gels were loaded for trichloroacetic acid ( T C A ) precipitable counts in o r d e r to adjust for variations in isotope injections The changes we r e p o r t here were seen consistently m all gels in whmh protein spots were clearly resolved. A f t e r sacrificing the animals (Nembutal, 50 mg/kg), the brains were r e m o v e d and examined grossly to evaluate the extent of the E C lesions (see Fig. 1). Only animals in which the lesions were judged to be at least 90% complete, and animals in which T C A precipltable counts from the hippocampus contralateral to the injection were at least four times higher than from frontal cortex (to ensure that the r a d m l a b e l e d protein in the hippocampus was from axonal transport), were used for the quantitative analysis. D a t a from seven of the ten ammals sacrificed 6 h following [35S]methionine injections are included in the quantitatxve analysis. Three 6-h animals were d r o p p e d from the analysis: one normal animal in which proteins were 'locally' labeled from b l o o d - b o r n e ra&oactivlty, and two experimental animals, one in which the injection of radiolabel was too deep (which resulted in a direct injection into the ipsilateral hippocampus), and one because of an incomplete lesion In the following discussion, ipsilateral and contralateral are used in reference to the [35S]methmnlne injections into the E C of one side. In lesioned animals, the hlppocampus contralateral to the injection sge is also the ~sprouting' hlppocampus, since it is ipsilateral to the EC lesion. Transport of radiolabeled proteins to the ipsilateral hlppocampus was detected at all time points tollowmg injection of [35S]methionine. Contralaterally, transported proteins could be detected as early as 3 h, but maximal labeling of fast-axonally transported proteins was not detected until 6 h post-injection A t 12 h, there was a de-
Lesion Site ( E n t o r h i n a l Cortex)
/
35S-methionine Injection ( E n t o r h i n a l Cortex)
| I
(Control)
Lesion Sate Fig 1 Schematic diagram showing the experimental paradigm In experimental animals, aspiration lesions were made of the right entorhmal cortex At the appropriate time following lesloning, the remaining intact entorhinal cortex was injected with [35S]methlonlne and the animals were allowed to survive for 4-6 or 15 days After sacrifice, the brains were removed and examined grossly to estimate the extent of the lesion The shaded area in this drawing denotes the extent of a typical lesion To ensure that proteins in the hlppocampus were labeled from axonal transport and not from blood- or CSF-borne label which would have resulted m locally labeled proteins, a piece of the frontal cortex was also removed and the proteins TCA-preclpltated Only animals in which the lesions were judged to be at least 90% complete and animals in which TCA-preclpltable counts from the hlppocampus were at least four times as great as those counted in frontal cortex were used for the quantitative analysis Note also that the [35S]methionlne lnjecnons were made into the non-sprouting hemisphere which means that the 'sprouting" hlppocampus could not have been labeled from direct injection or from diffusion of the isotope
crease m the density of protein spots labeled at 6 h and the appearance of new proteins suggesting some labeling of proteins by slow-axonal transport. Fig. 2 shows 2D autoradiographs of fast-axonally transported p r o t e m s to
149 TABLE I Transport o f newly synthestzed GAP-43 to the contralateral htppocampus m normal control ammals and to the 'sproutmg' hippocampus at 4-6 and 15 days followmg entorhmal cortex lestons
Newly synthesized proteins were labeled with [35S]methlonlne and subjected to 2D-PAGE autoradlography. Autoradlographs from normal control animals and ammals following unilateral aspiration of the EC were scanned and the protein spots quant~tated using a Mlcroscan 1000 gel analyzer (Technology Resources, Nashville, TN) Values expressed as '% GAP-43' represent densltometric values of total GAP-43 (all lsoforms) as a percent of the total rad~oactlv~ty of the autoradlograph minus background. The "GAP-431SNAP-25' column gives the ratio of the GAP-43 values relative to values determined on the same autoradlographs for SNAP-25 At 15 days post-lesion, SNAP-25 showed an approximate 1.3-fold upregulatlon. Values are shown corrected (no parentheses) or uncorrected (m parentheses) for SNAP-25 upregulation for the 15-day ammals. Regardless of what comparisons are made, GAP-43 expression shows no change untd 15 days following EC lesions when there is an approximate 2-fold increase m the transport of newly synthesized GAP-43 to the contralateral hlppocampus. When the GAP-43 values are normalized to the SNAP-25 protein values, the change at 15 days post-lesion Is highly significant (P < 0.003) Ammals
% GAP-43
GAP-43/SNAP-25
Controls
0.64 0 60
2.15 1 95
4-6 Days post-lesion
0.58 0 55
1.95 1.83
15 Days post-lesion
1.46 1.00 0.89
4.6 ~ ** 48 I 4.5
(4.4) (3.4) (3.0)
**P < 0.003 both hippocampi in normal control animals and to the contralateral or 'sprouting' hippocampus in lesioned animals 6 h following metabolic labeling. In the hippocampus of normal control animals, GAP-43 is resolved as two protein spots, indicating that there is a relatively high level of constitutive synthesis and transport of at least two GAP-43 isoforms in the perforant pathway. We have used quantitative densitometry (Microscan 1000, Technology Resources, Nashville, TN) to compare GAP-43 synthesis and transport levels in normal animals with its expression following EC lesions. Table I shows that transport of newly synthesized GAP-43 to the contralateral hippocampus is unchanged at 4-6 days following unilateral EC lesions, but is upregulated approximately 2-fold (100%) at 15 days post-lesion. In addition, at 15 days post-lesion, newly synthesized and transported GAP-43 can be resolved into at least three spots (Fig. 2D), representing different GAP-43 isoforms 39'4°. Although we were primarily interested in changes in GAP-43 synthesis and transport, our quantitative analysis also revealed consistent changes in the expression of two other acidic, fast-axonally transported proteins. One is a 25 kDa, developmentally regulated protein called
SNAP-25 (synaptosomal-associated protein or 'Superprotein '16) which is rich in methionine residues 34. While there appears to be little concensus about the regulation of this protein after axotomy 37'4z'43, SNAP-25 has been used by some investigators to normalize data when quantitating the upregulation of GAP-43 during growth states 11. In the present study, SNAP-25 showed an approximate 1.3-fold upregulation at 15 days post-lesion. Since we did not quantitate changes occurring at longer time points, it is possible that this does not represent the maximal upregulation in the expression of this protein. If SNAP-25 is associated with synaptogenesis or the stabilization of synaptic connections 35, there may be an increased requirement in the crossed perforant path as a result of the reactive synaptogenesis. Using GAP-43 values normalized to SNAP-25 protein density in the gels, the upregulation of GAP-43 at 15 days is highly significant (P < 0.003). The other fast axonally-transported protein which showed a consistent change in expression was an unknown protein of an approximate molecular weight of 70 kDa and approximate pI of 5.4. The synthesis and transport of this protein to the contralateral or 'sprouting' hippocampus was down-regulated at both 6 and 15 days following EC lesions (open arrow, Fig. 2B-D). The results of the present study suggest that in normal animals, the synthesis and transport of GAP-43 in the perforant pathway is significantly higher than in other pathways in the adult CNS. In all adult mammalian pathways or areas which have been examined, GAP-43 synthesis and/or transport are below detectable levels, or barely detectable in overexposed gels 17"t8'30'43. From knowing the radioactivity loaded onto the gels and the exposure times, we have been able to estimate that EC neurons normally synthesize and transport at least a 10fold greater amount of GAP-43 than is transported in adult rat optic nerve 12. It is also known from in situ hybridization studies that EC neurons are among a small set of cells which retain high levels of GAP-43 m R N A in adult animals ~°. Whereas message and protein levels are not always correlated (for example, for the c-fos protooncogene product Fos29), GAP-43 m R N A and protein levels do appear to be positively correlated at least in the systems which have been examined 2'6. Thus, EC neurons show an augmented synthesis of both GAP-43 message and protein, and the present study further indicates a relatively high constitutive level of transport of newly synthesized GAP-43 to presynaptic terminals in the dentate gyrus and hippocampus. The latter finding is consistent with our light microscopic immunolocalization showing that in the adult rat brain, GAP-43 is moderately concentrated within EC afferents projecting to the outer molecular layer of the dentate gyrus 26.
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Fig 2. Two-&menslonal autora&ographs showing the transport of newly synthesized m e m b r a n e protems to the (A) lpsllateral and (B) contralateral hlppocampus m a normal control ammal, and to the contralateral hlppocampus m ammals at 6 (C) and 15 (D) days following EC lesions. All ammals were sacrificed 6 h following metabohc labeling In A, some proteins may have been labeled from dlffUs]on of the [35S]methlonme to the lpsdateral hlppocampus In the h~ppocampus contralateral to the injeeuon ,lte ( B - D ) , all proteins represent fastaxonally transported proteins In lesioned animals. GAP-43 synthesis and transport appear to be unchanged when compared to control levels at 6 days, but are upregulated by 15 days The dlStrlbUt]on of GAP-43 between its different lsoforms is also changed following EC lesions By 6 days there is a shift m the relatwe a m o u n t of GAP-43 & s t n b u t e d between the two most acl&c lsoforms In a d d m o n , a basic lsoforrn is resolvable at 15 days SNAP-25 is a developmentally regulated methlonlne-nch fast-axonally transported protein whose expression is also upregulated at 15 days post-les~on The unlabeled protein indicated bv the open arrow was the only other protem which showed a reproduclble and consistent change m expression following unilateral EC lesions This protein showed a down-regulation at both 6 and 15 days post-lesion The acidic end of the gels is to the left (pl range = 3 5-10)
151 Following EC lesions, there is a loss of GAP-43 immunoreactivity from the outer denervated portion of the dentate molecular layer I°'3~ and a significant decrease in total GAP-43 levels in the ipsilateral dentate gyrus/hippocampus 2°'22. Moreover, the periods of initiation and growth of sprouting terminals In the denervated dentate gyrus are correlated with changes in the phosphorylation state of GAP-432°'22. The present results indicate that there is a 2-fold upregulation of the synthesis and transport of GAP-43 specifically to the terminals of EC neurons which are sprouting. From quantitative EM studies, we know that presynaptic terminal growth of EC afferents following unilateral EC lesions is robust by 6 days post-lesion 49. While some synapses are being added during this time, the major increases in synapse density are delayed relative to the period of maximal terminal growth 4Q. Thus, major increases in synapse formation begin between 8-10 days post-lesion and maximal levels are reached by approximately 15 days post-lesion, although synapse formation continues at a slow rate thereafter 49. Our quantitative data indicate that the increase in synthesis and transport of GAP-43 occurs between 6 and 15 days post-lesion, suggesting that sprouting or growth of presynaptic terminals results in a feedback upregulation in the expression of this specific protein. There is also other supportive data to suggest that changes in protein synthesis may be taking place at this time. Other studies have shown that layer II neurons in the surviving EC, which are the specific cells which sprout and reinnervate the outer part of the dentate molecular layer, show a significant increase in soma size at 8 days postlesion ~5. Hypertrophy of these cells is thought to reflect metabolic changes in the cell body related to an increased protein synthesis ~5. The upregulation and reaccumulation of GAP-43 within an increased number of terminals during sprouting is likely also to contribute to the trend of a return to normal GAP-43 levels as shown by Western blotting 2°'22 and the return of GAP-43 immunoreactivity in the denervated dentate gyrus as shown by immunocytochemistry 19"31. The latter findings are in agreement with studies in the PNS where terminal sprouting induced by treatment with insulin-like growth factor is correlated with elevated levels of GAP-43 within terminals in adult rat gluteus muscle, as shown by both immunocytochemistry and Western blotting 5. This increased expression in intramuscular nerve terminals during sprouting may also be due to a feedback upregulatmn in the synthesis and transport of GAP-43. During development and regeneration, GAP-43 synthesis and transport are elevated from 20- to 150-fold 12' 42.43. Presumably the elongation of axons over long distances which is necessary during such growth states
requires a significant and sustained upregulation of GAP-43 and other proteins involved in axon growth. In contrast, the local and limited growth of normal presynaptic terminals during lesion-induced sprouting and synaptogenesis does not appear to require a significant upregulation of GAP-43 synthesis and transport during the time of active presynaptic growth. In facL our results are consistent with the conclusion that for whatever immediate role GAP-43 plays in terminal proliferation, sufficient GAP-43 is present within presynaptic EC afferents. Within terminals where GAP-43 is concentrated, enough of the protein would be available to undergo rapid posttranslational modification, which may be directly related to presynaptic terminal growth or other modulatory events at these synapses. In support of this, we have shown that sprouting in the dentate gyrus, like LTP generated in the perforant pathway 24'25"38, is correlated with a change in the phosphorylation state of GAP-432°'22. In summary, we have found changes in the expression of at least three acidic fast-axonally transported proteins in the perforant pathway in response to unilateral EC lesions, We were particularly interested in changes which might occur in the synthesis and transport of GAP-43 during periods of terminal proliferation (4-6 days) and reactive synaptogenesis (15 days). Our results suggest that the 2-fold (100%) upregulation of GAP-43 synthesis and transport over the already high constitutive expression is likely to be due to a feedback regulation in response to the growth of presynaptic terminals. SNAP-25, which is also expressed at a high constitutive level in the perforant pathway, is also upregulated in response to EC lesions. The mechanism(s) responsible for the upregulation of GAP-43 and SNAP-25 are unknown. Entorhinal cortex neurons show high mRNA levels for both GAP43 l° and SNAP-25 a3. In the latter study 13, no increase in mRNA signal was reported for SNAP-25 following EC lesions, although this may be related to the resolution of in situ hybridization methods. For GAP-43, combined EC and fimbria fornix lesions have been reported to upregulate GAP-43 mRNA by 2- to 3-fold when Northern blotting is used 27. Accumulating data suggest also that GAP-43 synthesis and transport can be regulated, at least in part, by changes in GAP-43 mRNA stability6'2L 36. Our finding of a 2-fold change in the synthesis and transport of GAP-43 following EC lesions is certainly within a range which could be accounted for solely by a change in message stability. This, in turn, would suggest that factors affecting GAP-43 mRNA stability are being regulated during sprouting and reactive synaptogenesis and that this regulation may bring about the changes in the expression of GAP-43.
152 We would like to thank Ms Nancy Wall for help in running some of the gels, and Ms. Vera Atwood for typing the manuscript Coordinates for making medial entorhmal cortex injections were kindly
provided by Prot O Steward, University of Virginia This work was supported by NIH Grant NS25150 to J J N
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Bock, S. and Freeman, J.A., Possible role of GAP-43 in calcium regulatlon/neurotransmltter release, Ann. N Y A c i d Sct , 627 (1991) 75-93 Oyler, G A., Higgms, G . A , Hart, R A., Battenberg, E., Bdlingsley, M., Bloom, F.E and Wilson, M C., The Identification of a novel synaptosomal-assoclated protein, SNAP-25, differentially expressed by neuronal populations, J Cell Blol, 109 (1989) 3039-3052 Oyler, G.A , Polh, J.W., Wilson, M C. and Bllhngsley, M L , Developmental expression of the 25-kDa synaptosomal-associated protein (SNAP-25) m rat brain, Proc Natl. A c i d Sct USA, 88 (1991) 5247-5251 Perrone-Blzzozero, N.I., Irwin, N , Lewis, S . E , Fischer, I , Neve, R.L. and Benow~tz, L . I , Post-transcriptional regulation of GAP-43 mRNA levels during process outgrowth, Soc. Neurosct Abstr., 16 (1990) 814 Reh, T A., Redshaw, J.D and Blsby, M A , Axons of the pyramidal tract do not increase their transport of growth-associated proteins after axotomy, Mol Brain Res, 2 (1987) 1-6. Routtenberg, A , Lovmger, D.M and Steward, O., Selective increase in phosphorylatlon of a 47-kDA protein (FI) directly related to long-term potentiation, Behav NeuralBlol, 43 (1985) 3 Schreyer, D J and Skene, J.H P., Post-translational mo&ficat~on of a growth cone protein, GAP-43 an analysis using monoclonal antibodies, Soc Neuroscl Abstr, 13 (1987) 1480 Schreyer, D J. and Skene, J H P , Fate of GAP-43 in ascending spinal axons of D R G neurons after peripheral nerve injury delayed accumulation and correlation with regeneratwe potentml, J Neuroscl, 11 (1991) 3738-3751. Skene, J H . P , Axonal growth-associated proteins, Annu Rev Neurosct, 12 (1989) 127-156 Skene, J H P and Wlllard, M , Changes m axonally transported
43
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49
50
proteins during axon regeneration m toad retinal ganglion cells, J Cell Btol, 89 (1981a) 86-95 Skene, J.H P and Wlllard, M , Axonally transported protems associated with axon growth m rabbit central and peripheral nervous systems, J Cell Btol, 89 (1981b) 96-103 Skene, J.H.P, Jacobson, R.D., Stapes, G . J , McGmre, C B , Norden, J.J and Freeman, J A., A protein induced during nerve regeneration (GAP-43) is a major component of growth cone membranes, Science, 233 (1986) 783-786 Snipes, G J., Costello, B., McGulre, C B., Mayes, B N , Bock, S.S, Norden, J J. and Freeman, J A , Regulation of specific neuronal and non-neuronal proteins during development and following injury in the rat central nervous system. Prog. Brim Res, 71 (1987a) 155-175 Snipes, G J., Chen S Y., McGulre, C B., Costello, B R., Norden, J . J , Freeman, J A and Routtenberg, A , Evidence for the co-identification of GAP-43, a growth-associated protein, and FI, a plasticity-associated protein, J Neurosct, 7 (1987b) 4066-4075 Steward, O., Remnervatlon of dentate gyrus by homologous afferents following entorhinal cortical lesions in adult rats, Science, 194 (1976) 426-428 Steward, O and Vmsant, S L , Identification of the cells of orIgin of a central pathway which sprouts following lesions m mature rats, Brim Res, 147 (1978) 223-243. Steward, O. and Vlnsant, S . L , The process of remnervation m the dentate gyrus of the adult rat' a quantitative electron microscopic analysis of terminal prohferation and reactive synaptogenesis, J Comp Neurol, 214 (1983) 370-386 Waklm, B T , Alexander, K A . Masure, H R , Cimler, B M., Storm, D R and Walsh, K A., Amino acid sequence of P57, a neurospeclfic calmoduhn-blndIng protein, Biochemistry, 26 (1987) 7466-7470
147
Elsewer BRESM 80128
Synthesis and transport of GAP-43 in entorhinal cortex neurons and perforant pathway during lesion-induced sprouting and reactive synaptogenesis Li-Hsien Lin, Susan Bock, Kenneth Carpenter, Michael Rose and Jeanette J. Norden Department of Cell Biology, Vanderbdt Universtty Medwal School, Nashvdle, TN 37232 (USA)
(Accepted 11 February 1992) Key words" Growth-associated protein; Synaptlc plasticity; Hlppocampus; Axonal transport; Synaptosomal-assocmtedprotein
Metabohc labehng and quantitative 2D gel autoradlography were used to assess changes m the synthes~s and transport of GAP-43 m entorhmal cortex (EC) neurons and perforant pathway during lesion-reduced sprouting and reactive synaptogenesls. In normal adult rats, there Is a high constltutwe level of GAP-43 synthesis and transport m EC neurons projecting to the hlppocampus Following umlateral EC lesions, there is a 2-fold (100%) increase m the transport of newly synthesized GAP-43 to the contralateral or 'sprouting" hippocampus. The txmmg of this upregulatlon (between 6 and 15 days) suggests that changes in GAP-43 expression occur m response to the growth of presynaptlc terminals during sprouting Growth-associated protein-43 (GAP-43; B-50:7; Fl46; pp4628; neuromodulin 5°) is a fast-axonally transported phosphoprotein postulated to play a role in axon growth and synaptlc plasticity (reviewed in refs. 3,12,23,32,33, 41,45). During development 12n8'43 and regeneration 1'42, axon growth is correlated with a significant upregulation of GAP-43 synthesis and transport. Consistent with these findings, GAP-43 is localized to fiber pathways in the developing brain 26 and concentrated in growth cones 9'44. In adult animals, GAP-43 is concentrated in axon terminals 4'26, particularly within so-called 'plastic' areas such as the neocortex, cerebellum and hippocampus 4'z6, where it is believed to play a role in synaptic plasticity. For example, in the hippocampus, the posttranslational modification of GAP-43 has been linked to changes in the efficacy of synapses. In long-term potentiation (LTP), there is a significant increase in the in vitro phosphorylation of GAP-43 (F146) specifically linked to the maintenance of LTP z4'25'38 Our lab has been interested in examining the role GAP-43 may play in another form of synaptic plasticity in the adult brain. This type of plasticity, which is termed lesioned-induced sprouting and reactive synaptogenesis, involves the terminal growth of normal axons in response to the removal of other afferents and is particularly robust in the hippocampus (reviewed in refs. 7,8). For example, in adult rats there is a major projection of the entorhinal cortex (EC) to the outer 2/3-3/4 of the
molecular layer of the ipsilateral dentate gyrus and a very minor projection to the contralateral side 47. Following unilateral removal of the EC, the normally sparse contralateral EC projection, commissural/associational fibers, and other normal afferents, 'sprout' axon terminals and form new synapses in the denervated portion of the dentate molecular layer 47. Although the 'sprouting' paradigm is widely used to study synaptic plasticity, only one study has addressed the issue of changes in presynaptic protein synthesis and transport during this process. Using light-microscopic autoradiography to visualize all 3H-labeled proteins transported in commissural/ associational afferents following EC lesions, Goldowitz and Cotman 14 found no change in the labeling pattern of fast-axonally transported proteins during sprouting. While the overall labeling pattern of proteins might not change during sprouting and reactive synaptogenesis, the expression of individual proteins may be regulated during these processes. Here we have re-examined this question by quantitating changes in the expression of a specific protein which is postulated to play a role in both axon growth and synaptic plasticity. Using 2D gel autoradiography we have quantitated changes over a timecourse in the synthesis and transport of GAP-43 in EC neurons and perforant pathway during terminal sprouting (4-6 days) and reactive synaptogenesis (15 days). Sixteen adult male Sprague-Dawley rats (250-330 g) were anesthetized with ketamine (87 mg/kg) and xyla-
Correspondence J J Norden, Department of Cell Biology, Vanderbllt Umversity Me&cal School. Nashvdle. TN 37232-2175, USA Fax: (1)
(615) 343-4539
148 zme (13 mg/kg). In normal (unlesioned) ammals, the E C was exposed on one u d e and injecnons of 0,5 #1 of [35S]methionine were m a d e into the medial E C 44 with a Hamilton microliter syringe. Injections were made over a 2-rain period and the syringe was left in place for an additional 10 min Animals were sacrificed by drug overdose (Nembutal; 50 mg/kg) at 2, 3, 6 or 12 h following the last injection. Both dorsal hlppocampi were removed, and high-speed m e m b r a n e fractions were p r e p a r e d and subjected to 2 D - P A G E autoradiography as described previously 12. F r o m these data in normal animals, we determined that 6 h was the optimal time for labehng of proteins to the contralateral hippocampus by fast-axonal transport. In experimental ammals, the right EC was removed by aspiratmn, and the animals allowed to survive for 4 - 6 or 15 days. A t the appropriate time, the animal was re-anesthetized, [35S]methionlne rejections were made into the remaining intact EC, and the dorsal hippocampi and dentate gyrl were r e m o v e d 6 h later and subjected to 2 D - P A G E autoradiography. Gels were loaded for trichloroacetic acid ( T C A ) precipitable counts in o r d e r to adjust for variations in isotope injections The changes we r e p o r t here were seen consistently m all gels in whmh protein spots were clearly resolved. A f t e r sacrificing the animals (Nembutal, 50 mg/kg), the brains were r e m o v e d and examined grossly to evaluate the extent of the E C lesions (see Fig. 1). Only animals in which the lesions were judged to be at least 90% complete, and animals in which T C A precipltable counts from the hippocampus contralateral to the injection were at least four times higher than from frontal cortex (to ensure that the r a d m l a b e l e d protein in the hippocampus was from axonal transport), were used for the quantitative analysis. D a t a from seven of the ten ammals sacrificed 6 h following [35S]methionine injections are included in the quantitatxve analysis. Three 6-h animals were d r o p p e d from the analysis: one normal animal in which proteins were 'locally' labeled from b l o o d - b o r n e ra&oactivlty, and two experimental animals, one in which the injection of radiolabel was too deep (which resulted in a direct injection into the ipsilateral hippocampus), and one because of an incomplete lesion In the following discussion, ipsilateral and contralateral are used in reference to the [35S]methmnlne injections into the E C of one side. In lesioned animals, the hlppocampus contralateral to the injection sge is also the ~sprouting' hlppocampus, since it is ipsilateral to the EC lesion. Transport of radiolabeled proteins to the ipsilateral hlppocampus was detected at all time points tollowmg injection of [35S]methionine. Contralaterally, transported proteins could be detected as early as 3 h, but maximal labeling of fast-axonally transported proteins was not detected until 6 h post-injection A t 12 h, there was a de-
Lesion Site ( E n t o r h i n a l Cortex)
/
35S-methionine Injection ( E n t o r h i n a l Cortex)
| I
(Control)
Lesion Sate Fig 1 Schematic diagram showing the experimental paradigm In experimental animals, aspiration lesions were made of the right entorhmal cortex At the appropriate time following lesloning, the remaining intact entorhinal cortex was injected with [35S]methlonlne and the animals were allowed to survive for 4-6 or 15 days After sacrifice, the brains were removed and examined grossly to estimate the extent of the lesion The shaded area in this drawing denotes the extent of a typical lesion To ensure that proteins in the hlppocampus were labeled from axonal transport and not from blood- or CSF-borne label which would have resulted m locally labeled proteins, a piece of the frontal cortex was also removed and the proteins TCA-preclpltated Only animals in which the lesions were judged to be at least 90% complete and animals in which TCA-preclpltable counts from the hlppocampus were at least four times as great as those counted in frontal cortex were used for the quantitative analysis Note also that the [35S]methionlne lnjecnons were made into the non-sprouting hemisphere which means that the 'sprouting" hlppocampus could not have been labeled from direct injection or from diffusion of the isotope
crease m the density of protein spots labeled at 6 h and the appearance of new proteins suggesting some labeling of proteins by slow-axonal transport. Fig. 2 shows 2D autoradiographs of fast-axonally transported p r o t e m s to
149 TABLE I Transport o f newly synthestzed GAP-43 to the contralateral htppocampus m normal control ammals and to the 'sproutmg' hippocampus at 4-6 and 15 days followmg entorhmal cortex lestons
Newly synthesized proteins were labeled with [35S]methlonlne and subjected to 2D-PAGE autoradlography. Autoradlographs from normal control animals and ammals following unilateral aspiration of the EC were scanned and the protein spots quant~tated using a Mlcroscan 1000 gel analyzer (Technology Resources, Nashville, TN) Values expressed as '% GAP-43' represent densltometric values of total GAP-43 (all lsoforms) as a percent of the total rad~oactlv~ty of the autoradlograph minus background. The "GAP-431SNAP-25' column gives the ratio of the GAP-43 values relative to values determined on the same autoradlographs for SNAP-25 At 15 days post-lesion, SNAP-25 showed an approximate 1.3-fold upregulatlon. Values are shown corrected (no parentheses) or uncorrected (m parentheses) for SNAP-25 upregulation for the 15-day ammals. Regardless of what comparisons are made, GAP-43 expression shows no change untd 15 days following EC lesions when there is an approximate 2-fold increase m the transport of newly synthesized GAP-43 to the contralateral hlppocampus. When the GAP-43 values are normalized to the SNAP-25 protein values, the change at 15 days post-lesion Is highly significant (P < 0.003) Ammals
% GAP-43
GAP-43/SNAP-25
Controls
0.64 0 60
2.15 1 95
4-6 Days post-lesion
0.58 0 55
1.95 1.83
15 Days post-lesion
1.46 1.00 0.89
4.6 ~ ** 48 I 4.5
(4.4) (3.4) (3.0)
**P < 0.003 both hippocampi in normal control animals and to the contralateral or 'sprouting' hippocampus in lesioned animals 6 h following metabolic labeling. In the hippocampus of normal control animals, GAP-43 is resolved as two protein spots, indicating that there is a relatively high level of constitutive synthesis and transport of at least two GAP-43 isoforms in the perforant pathway. We have used quantitative densitometry (Microscan 1000, Technology Resources, Nashville, TN) to compare GAP-43 synthesis and transport levels in normal animals with its expression following EC lesions. Table I shows that transport of newly synthesized GAP-43 to the contralateral hippocampus is unchanged at 4-6 days following unilateral EC lesions, but is upregulated approximately 2-fold (100%) at 15 days post-lesion. In addition, at 15 days post-lesion, newly synthesized and transported GAP-43 can be resolved into at least three spots (Fig. 2D), representing different GAP-43 isoforms 39'4°. Although we were primarily interested in changes in GAP-43 synthesis and transport, our quantitative analysis also revealed consistent changes in the expression of two other acidic, fast-axonally transported proteins. One is a 25 kDa, developmentally regulated protein called
SNAP-25 (synaptosomal-associated protein or 'Superprotein '16) which is rich in methionine residues 34. While there appears to be little concensus about the regulation of this protein after axotomy 37'4z'43, SNAP-25 has been used by some investigators to normalize data when quantitating the upregulation of GAP-43 during growth states 11. In the present study, SNAP-25 showed an approximate 1.3-fold upregulation at 15 days post-lesion. Since we did not quantitate changes occurring at longer time points, it is possible that this does not represent the maximal upregulation in the expression of this protein. If SNAP-25 is associated with synaptogenesis or the stabilization of synaptic connections 35, there may be an increased requirement in the crossed perforant path as a result of the reactive synaptogenesis. Using GAP-43 values normalized to SNAP-25 protein density in the gels, the upregulation of GAP-43 at 15 days is highly significant (P < 0.003). The other fast axonally-transported protein which showed a consistent change in expression was an unknown protein of an approximate molecular weight of 70 kDa and approximate pI of 5.4. The synthesis and transport of this protein to the contralateral or 'sprouting' hippocampus was down-regulated at both 6 and 15 days following EC lesions (open arrow, Fig. 2B-D). The results of the present study suggest that in normal animals, the synthesis and transport of GAP-43 in the perforant pathway is significantly higher than in other pathways in the adult CNS. In all adult mammalian pathways or areas which have been examined, GAP-43 synthesis and/or transport are below detectable levels, or barely detectable in overexposed gels 17"t8'30'43. From knowing the radioactivity loaded onto the gels and the exposure times, we have been able to estimate that EC neurons normally synthesize and transport at least a 10fold greater amount of GAP-43 than is transported in adult rat optic nerve 12. It is also known from in situ hybridization studies that EC neurons are among a small set of cells which retain high levels of GAP-43 m R N A in adult animals ~°. Whereas message and protein levels are not always correlated (for example, for the c-fos protooncogene product Fos29), GAP-43 m R N A and protein levels do appear to be positively correlated at least in the systems which have been examined 2'6. Thus, EC neurons show an augmented synthesis of both GAP-43 message and protein, and the present study further indicates a relatively high constitutive level of transport of newly synthesized GAP-43 to presynaptic terminals in the dentate gyrus and hippocampus. The latter finding is consistent with our light microscopic immunolocalization showing that in the adult rat brain, GAP-43 is moderately concentrated within EC afferents projecting to the outer molecular layer of the dentate gyrus 26.
150
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Fig 2. Two-&menslonal autora&ographs showing the transport of newly synthesized m e m b r a n e protems to the (A) lpsllateral and (B) contralateral hlppocampus m a normal control ammal, and to the contralateral hlppocampus m ammals at 6 (C) and 15 (D) days following EC lesions. All ammals were sacrificed 6 h following metabohc labeling In A, some proteins may have been labeled from dlffUs]on of the [35S]methlonme to the lpsdateral hlppocampus In the h~ppocampus contralateral to the injeeuon ,lte ( B - D ) , all proteins represent fastaxonally transported proteins In lesioned animals. GAP-43 synthesis and transport appear to be unchanged when compared to control levels at 6 days, but are upregulated by 15 days The dlStrlbUt]on of GAP-43 between its different lsoforms is also changed following EC lesions By 6 days there is a shift m the relatwe a m o u n t of GAP-43 & s t n b u t e d between the two most acl&c lsoforms In a d d m o n , a basic lsoforrn is resolvable at 15 days SNAP-25 is a developmentally regulated methlonlne-nch fast-axonally transported protein whose expression is also upregulated at 15 days post-les~on The unlabeled protein indicated bv the open arrow was the only other protem which showed a reproduclble and consistent change m expression following unilateral EC lesions This protein showed a down-regulation at both 6 and 15 days post-lesion The acidic end of the gels is to the left (pl range = 3 5-10)
151 Following EC lesions, there is a loss of GAP-43 immunoreactivity from the outer denervated portion of the dentate molecular layer I°'3~ and a significant decrease in total GAP-43 levels in the ipsilateral dentate gyrus/hippocampus 2°'22. Moreover, the periods of initiation and growth of sprouting terminals In the denervated dentate gyrus are correlated with changes in the phosphorylation state of GAP-432°'22. The present results indicate that there is a 2-fold upregulation of the synthesis and transport of GAP-43 specifically to the terminals of EC neurons which are sprouting. From quantitative EM studies, we know that presynaptic terminal growth of EC afferents following unilateral EC lesions is robust by 6 days post-lesion 49. While some synapses are being added during this time, the major increases in synapse density are delayed relative to the period of maximal terminal growth 4Q. Thus, major increases in synapse formation begin between 8-10 days post-lesion and maximal levels are reached by approximately 15 days post-lesion, although synapse formation continues at a slow rate thereafter 49. Our quantitative data indicate that the increase in synthesis and transport of GAP-43 occurs between 6 and 15 days post-lesion, suggesting that sprouting or growth of presynaptic terminals results in a feedback upregulation in the expression of this specific protein. There is also other supportive data to suggest that changes in protein synthesis may be taking place at this time. Other studies have shown that layer II neurons in the surviving EC, which are the specific cells which sprout and reinnervate the outer part of the dentate molecular layer, show a significant increase in soma size at 8 days postlesion ~5. Hypertrophy of these cells is thought to reflect metabolic changes in the cell body related to an increased protein synthesis ~5. The upregulation and reaccumulation of GAP-43 within an increased number of terminals during sprouting is likely also to contribute to the trend of a return to normal GAP-43 levels as shown by Western blotting 2°'22 and the return of GAP-43 immunoreactivity in the denervated dentate gyrus as shown by immunocytochemistry 19"31. The latter findings are in agreement with studies in the PNS where terminal sprouting induced by treatment with insulin-like growth factor is correlated with elevated levels of GAP-43 within terminals in adult rat gluteus muscle, as shown by both immunocytochemistry and Western blotting 5. This increased expression in intramuscular nerve terminals during sprouting may also be due to a feedback upregulatmn in the synthesis and transport of GAP-43. During development and regeneration, GAP-43 synthesis and transport are elevated from 20- to 150-fold 12' 42.43. Presumably the elongation of axons over long distances which is necessary during such growth states
requires a significant and sustained upregulation of GAP-43 and other proteins involved in axon growth. In contrast, the local and limited growth of normal presynaptic terminals during lesion-induced sprouting and synaptogenesis does not appear to require a significant upregulation of GAP-43 synthesis and transport during the time of active presynaptic growth. In facL our results are consistent with the conclusion that for whatever immediate role GAP-43 plays in terminal proliferation, sufficient GAP-43 is present within presynaptic EC afferents. Within terminals where GAP-43 is concentrated, enough of the protein would be available to undergo rapid posttranslational modification, which may be directly related to presynaptic terminal growth or other modulatory events at these synapses. In support of this, we have shown that sprouting in the dentate gyrus, like LTP generated in the perforant pathway 24'25"38, is correlated with a change in the phosphorylation state of GAP-432°'22. In summary, we have found changes in the expression of at least three acidic fast-axonally transported proteins in the perforant pathway in response to unilateral EC lesions, We were particularly interested in changes which might occur in the synthesis and transport of GAP-43 during periods of terminal proliferation (4-6 days) and reactive synaptogenesis (15 days). Our results suggest that the 2-fold (100%) upregulation of GAP-43 synthesis and transport over the already high constitutive expression is likely to be due to a feedback regulation in response to the growth of presynaptic terminals. SNAP-25, which is also expressed at a high constitutive level in the perforant pathway, is also upregulated in response to EC lesions. The mechanism(s) responsible for the upregulation of GAP-43 and SNAP-25 are unknown. Entorhinal cortex neurons show high mRNA levels for both GAP43 l° and SNAP-25 a3. In the latter study 13, no increase in mRNA signal was reported for SNAP-25 following EC lesions, although this may be related to the resolution of in situ hybridization methods. For GAP-43, combined EC and fimbria fornix lesions have been reported to upregulate GAP-43 mRNA by 2- to 3-fold when Northern blotting is used 27. Accumulating data suggest also that GAP-43 synthesis and transport can be regulated, at least in part, by changes in GAP-43 mRNA stability6'2L 36. Our finding of a 2-fold change in the synthesis and transport of GAP-43 following EC lesions is certainly within a range which could be accounted for solely by a change in message stability. This, in turn, would suggest that factors affecting GAP-43 mRNA stability are being regulated during sprouting and reactive synaptogenesis and that this regulation may bring about the changes in the expression of GAP-43.
152 We would like to thank Ms Nancy Wall for help in running some of the gels, and Ms. Vera Atwood for typing the manuscript Coordinates for making medial entorhmal cortex injections were kindly
provided by Prot O Steward, University of Virginia This work was supported by NIH Grant NS25150 to J J N
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