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Axotomy Increases the Expression of Glucose-Regulated Protein 78 kDa in Rat Facial Nucleus
EXPERIMENTAL NEUROLOGY ARTICLE NO.
146, 10–16 (1997)
EN976526
Axotomy Increases the Expression of Glucose-Regulated Protein 78 kDa in Rat Facial Nucleus M. Teresa Moreno-Flores,1 Ursula E. Olaza´bal, and Georg W. Kreutzberg2 Max-Planck-Institute of Psychiatry, Department of Neuromorphology, Am Klopferspitz 18a, 82152 Martinsried, Germany
supplying energy during axonal reaction and regeneration have been described (9, 16, 22, 24). In facial and hypoglossal motor nuclei, there is an increase in glucose utilization and protein synthesis after axotomy of their respective nerves (9, 22, 24), which was found as early as 24 h postaxotomy and persisted after the functional recovery. A variety of stressful conditions are known to induce the expression of heat shock proteins (hsp) (6). Stress proteins include the glucose-regulated protein (GRP) family. GRPs share high homology with some hsps, are localized in endoplasmic reticulum (ER) (5, 14, 15), are implicated in cellular protein folding and subunit assembly, and are induced under conditions of glucose starvation (2–4, 19, 23). In addition, GRPs are induced by amino acid analogs, as are hsps, and by GRP-selective stressors, such as glycosylation inhibitors and Ca21 ionophores (6, 18, 20, 21). Although information is available on early postaxotomy glucose utilization and protein synthesis, the cellular mechanisms underlying these responses are not clearly understood. The present studies were, therefore, carried out to determine the effect of axotomy on the expression and regulation of GRP 78 kDa (GRP78) in the facial nucleus (FN).
Nerve injuries lead to metabolic and morphological changes in the cell bodies of the neurons of origin. Increases in glucose turnover in axotomized facial and hypoglossal motor nuclei have been described. Glucoseregulated protein 78 kDa (GRP78) is implicated in cellular protein folding and subunit assembly and responds to glucose deficiency. We performed Western blot and immunohistochemistry to determine the effect of axotomy on the expression and regulation of GRP78 in the facial nucleus (FN). Facial nerve axotomy caused a larger and longer increase of GRP78 in the ipsilateral FN than in the contralateral FN. In right ipsilateral FN, axotomy resulted in elevation of GRP78 protein levels, first detected at 12 h and which reached significant, maximal induction at 24 h (75 6 27% increase). GRP78 protein levels decreased at later time points, but remained elevated over sham-operated controls. In contrast, no significant increase in GRP78 concentrations was found in contralateral left FN. Immunocytochemically, positive GRP78 staining was found mainly in the cytoplasm of motoneurons; there was no nuclear staining. Prominent GRP78-immunostaining appeared in axotomized motoneurons at 24 h postaxotomy as compared with the contralateral, unoperated controls. This augmentation was also observed at 4 and 7 days postaxotomy. The possibility that glucose metabolism and GRP78 levels are two parallel events in the injured facial nucleus is discussed. r 1997
MATERIALS AND METHODS
Animals and Surgery
Academic Press
A total of 63 Wistar male rats (250–300 g) were used in biochemical and morphological studies. The animals were housed in small groups on a 12:12 light:dark cycle and provided with food and water ad libitum. Axotomy time course studies were done by transecting the right facial nerve at the stylomastoid foramen under ether anesthesia. For biochemical studies, animals were sacrificed at 6 h (n 5 4), 12 h (n 5 10), 24 h (n 5 8), 72 h (3 days; n 5 10), 168 h (7 days; n 5 6), or 336 h (14 days; n 5 6) following axotomy by brief exposure to CO2 (30 s). The facial nuclei (FN) (right, operated, ipsilateral to the transection; left, unoperated, contralateral to the transection) were microdissected from a 2-mm slice and stored at 270°C. Three rats were used as shamoperated controls (S/O; exposing facial nerve without
INTRODUCTION
Axotomy and other nerve injuries lead to metabolic and morphological changes in the cell bodies of the neurons of origin (11). In the peripheral nervous system, nerve regeneration and functional restoration usually follow these processes. Metabolic changes in RNA, protein synthesis, and in oxidative metabolism
1 Present address: Centro de Biologı´a Molecular ‘‘Severo Ochoa’’ (CSIC-UAM), Facultad de Ciencias, Universidad Auto´noma, 28049 Madrid, Spain. Fax: (00341) 397 48 70. 2 Reprint requests to Georg W. Kreutzberg.
0014-4886/97 $25.00 Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.
10
AXOTOMY REGULATION OF GRP78 IN FACIAL NUCLEUS
transection; survival time 24 h) and two additional rats were used as unoperated ‘‘intact’’ controls. In morphological studies, animals were operated as described and sacrificed at 6 h (n 5 2), 24 h (n 5 5), 4 days (n 5 3), 7 days (n 5 2), or 15 days (n 5 2) later by trans-cardial perfusion with isotonic saline solution (0.9% NaCl) for 10 min under deep chloral hydrate anesthesia (30% solution, 50 mg/kg body weight). The brains were removed, immediately frozen, and stored at 270°C until ready to use. Materials Immunological studies were done using two different antibodies directed against GRP78 protein: (i) A mouse monoclonal anti-GRP78 antibody (StressGen, B.C., Canada), prepared against an heptapeptide from the carboxy terminus of rat GRP78. The antibody identified primarily GRP78, but also crossreacted with another glucose stress protein, GRP 94 kDa. (ii) A rabbit polyclonal anti-GRP78 (StressGen), prepared against a synthetic peptide from the carboxy terminus of human GRP78. This antibody did not show cross-reactivity with other proteins. Pure hsp70 protein was also obtained from StressGen. Polyvinyldifluoride membranes were obtained from Schleicher & Schuell. For immunoblotting, secondary antibodies and enhanced chemiluminescence kits were obtained from Amersham. Secondary antibodies used in immunocytochemical studies, as well as normal sera and avidin–biotin Vectastain kits, were purchased from Vector Labs. Biochemical Studies Electrophoresis. Extracted facial nuclei were prepared in sodium dodecyl sulfate (SDS) sample buffer, containing 0.5 M Tris base, 0.1 M EDTA, 2% SDS, 10% glycerol, and 10% mercaptoethanol and heat denatured. Samples were normalized with respect to total protein concentration (1) and 10 µg protein from each were resolved on two-phase one-dimensional gels consisting of a 4% acrylamide upper stacking gel and a 10% acrylamide lower resolving gel (6 3 9 cm) (10), using a Hoeffer SE 215 mini gel apparatus. On each gel, single samples from the entire axotomy time course (S/O, 6 h, 12 h, 24 h, 3 days, 7 days, and 14 days) for right (operated) or left (control) nuclei were resolved. Thus, every gel represented an individual time course analysis for right and/or left FN. A total of 19 gels were electrophoresed at constant voltage (150 V) and time (1:15 h), which comprised two independent experiments. Western blots/enhanced chemiluminescence. Proteins were captured on polyvinyldifluoride filters. Immunoblots were blocked in Tween buffer (0.01 M Tris base, 0.5 M NaCl, 0.5% Tween 20) containing 10% nonfat dry milk and incubated in the monoclonal anti-GRP78 antibody (1:500). A second blocking step in 5% normal
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serum was done, followed by incubation in the horseradish peroxidase-conjugated secondary antibody. Immunoblots were washed overnight in Tween buffer prior to developing with enhanced chemiluminescence reagent. Specific GRP78 bands were detected on film using enhanced chemiluminescence and exposing to film at various times to optimize signal visualization. These parameters were then maintained constant for all blots thereafter. Specific bands were digitized and analyzed using a laser scanner equipped with Image Quant analysis software (Molecular Dynamics, CA). Once bands were defined, optical densities (OD) units of signal over background were recorded and averaged across blots. Results are expressed as mean OD units (GRP78 immunoreactivity) 6 standard error (SEM) for every experimental condition. Further, the mean and coefficient of variance were calculated for every experimental condition and compared to controls. If the two mean values differed more than twice the coefficient of variance, these values were additionally compared using an unpaired two-tailed student’s t test. Immunohistochemical Studies The cellular localization of GRP78 protein was examined by immunohistochemistry, to determine the cell types in FN which could be involved in the response of this protein to axotomy. Briefly, fresh 20-µm cryosections containing the facial nuclei were incubated in a 1:200 dilution of the primary anti-GRP78 antibody (monoclonal or polyclonal) followed by incubation in the biotinylated secondary antibody (for each corresponding primary antibody). Sections were immunostained using the Vectastain ABC kit (Vector Lab.) and developed in diaminobenzidine/H2O2, dehydrated, coverslipped, and examined using light microscopy. Control Conditions Since GRP78 and hsp70 share approximately 60% homology (14, 15), it was important to determine the specificity of the GRP78 antibody. Preadsorption control experiments were carried out on alternate sections from 1- and 4-day postaxotomy animals by first incubating pure hsp70 protein with the primary anti-grp78 antibody and then applying to tissue sections. Additional controls were also involved omitting the primary GRP78 antibody from the immunostaining procedure. RESULTS
Effects of Axotomy on GRP78 Protein Levels: Western Blots Intact and sham-operated animals were compared to establish control levels of GRP78 in FN. We found differences in the baseline for right and left FN, both in intact and sham-operated animals. For control intact
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´ BAL, AND KREUTZBERG MORENO-FLORES, OLAZA
animals, the left side had a mean of 171% with respect to the right FN that represented 100% in values of optical density units. For control sham-operated animals the mean was 130% for the left FN, contralateral to the facial nerve exposed in the surgery without transection, with respect to 100% values of right/
FIG. 2. Percentage of control increase in GRP78 immunoreactivity in right (axotomized; filled boxes) and left (unoperated; unfilled boxes) facial nucleus at different times postaxotomy, in respect to their controls. In single immunoblots (right or left), percentage increases over controls were determined for each experimental condition and pooled across blots from two independent experiments. Results are expressed as mean percentage increases 6 standard error.
FIG. 1. (A) Time course induction of GRP78 protein in the right facial nucleus in response to axotomy. Sample preparation and Western blot analysis using anti-grp78 monoclonal antibody was done as described under Materials and Methods. 0 hours represents the sham-operated control rats in which the right facial nerve was exposed but not transected. At each time point, the results are expressed as mean optical density units 6 standard error. Right FN axotomy caused a significant rise in GRP78 concentrations, which was maximal at 24 h (P 5 0.0007, unpaired two-tailed t test). (B) GRP78 immunoreactivity in the left facial nucleus after axotomy of the contralateral facial nerve. Time course studies were carried out as for right FN and the results are expressed as mean optical density units 6 standard error for every experimental condition. Following right FN transection, an increase in GRP78 levels were observed at 12 h, but did not significantly differ from sham-op controls (P 5 0.22; unpaired two-tailed t test). Concentrations decreased at 24 h and then returned to baseline. FN, facial nucleus; GRP78, glucoseregulated protein 78 kDa.
ipsilateral FN. Therefore the differences in the baseline for both FN were not due to surgery stress. Means 6 SEM at each time point for right or left FN are shown in Fig. 1, representative of two separate independent experiments. In right FN, axotomy resulted in an elevation of GRP78 protein levels, first detected at 12 h and which reached maximal induction at 24 h (P 5 0.0007; unpaired, two-tailed t test; Fig. 1A). This induction, at 24 h, represented an 75 6 27% increase and was consistent in 7 of 7 rats evaluated, as shown in Fig. 2. These data are the pooled values of two independent experiments. Due to the uniformity in the GRP78 response in operated FN for all animals studied at this time point, the standard error of the mean value at this time point was consistently small across independent experiments. Similarly, the variation observed for S/O right FN was also small since no change in GRP78 levels were found between animals. GRP78 protein levels decreased at 3 days postaxotomy and at later time points, but remained elevated over S/O controls. The corresponding variations were also higher. In contrast, in the corresponding contralateral FN, maximal levels were observed at 12 h postaxotomy (Figs. 1B and 2; 39 6 11%; P 5 0.22; n 5 9), which decreased at 24 h (19 6 10%; n 5 7; Fig. 2) and returned to baseline levels thereafter. Notably, the response at 12 h in the unoperated FN was less pronounced that in the operated, right nucleus (Fig. 2; 39 6 11% vs 65 6 21%), was not significant and was transient, in comparison with the operated side in which GRP78 response peaked at 24 h and maintained higher than the baseline for 14 days. Therefore, GRP78 concentrations in left FN varied in response to axotomy of the contralateral facial nerve, but insignificantly.
AXOTOMY REGULATION OF GRP78 IN FACIAL NUCLEUS
FIG. 3. Single Western blot analysis of axotomy time course effects on GRP78 protein in (A) right facial nucleus and (B) left facial nucleus. (A) In right axotomized facial nucleus, GRP78-specific band intensity is greater at 12 h versus control (c.f. lanes 1 and 3), peaks at 24 h (lane 4), and begins to decrease at 72 h, yet remaining higher than control (c.f. lanes 1, 5, 6, and 7). (B) GRP78 band immunoreactivity in left, unoperated facial nucleus transiently increased at 12 h after axotomy of the right facial nerve (lane 3) and returned to baseline thereafter.
Individual Western blot experiments from right (A, axotomized) and left (B, unoperated) FN are shown in Fig. 3. These are representative cases of the pooled data for each analysis. Immunoreactive bands represent tissue sampled from independent animals and processed as described under Materials and Methods. In single immunoblots, intensely reactive bands at 78 kDa were observed in all lanes. In right FN (Fig. 3A, top), postaxotomy GRP78 increases were first detectable at 12 h (lane 3). Immunoreactive band intensity was maximal at 24 h (lane 4), being markedly higher than in S/O controls or 6 h postaxotomy (c.f. lanes 1, 2, and 4). GRP78 protein concentrations decreased at 3 days, but were nevertheless elevated over the postaxotomy time course tested (lanes 5, 6, and 7). In single, left FN immunoblots (Fig. 3B, bottom), a transient rise in GRP78 protein was noted at 12 h after axotomy of the contralateral nerve (c.f. lanes 1 and 3), which later returned to control levels (lanes 4–7). GRP78 Immunohistochemistry Both monoclonal and polyclonal antibodies presented similar patterns of immunostaining across all animals under the conditions tested. In all sections, these antibodies stained primarily neurons and appeared as either diffuse or punctate in the cytoplasmic space; there was no nuclear staining (Fig. 4). In FN, GRP78immunoreactivity appeared in motoneurons. Shortly following axotomy (6 h), no detectable changes in immunostaining of motoneurons were found (not shown). However, prominent GRP78-immunostaining of right FN motoneurons appeared at 24 h postaxotomy (Fig. 4B), as compared with the contralateral, unoperated FN (Fig. 4A). This augmentation was detected in all animals studied at this time point and was consis-
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tent with that observed in the Western blot analysis (Fig. 1). A slight increase in staining intensity of the neuropil in operated FN could be also seen. Similar increases in motoneuron immunostaining were also observed at 4 days (Fig. 4C, left FN, and 4D, right FN) and 7 days (Fig. 4E, left FN, and 4F, right FN) after transection, except that higher individual staining variability was noted and thus resulting in less marked differences in right–left FN comparisons. These findings were also noted at the level of immunoblotting (Fig. 1). In preadsorption control experiments, positive GRP78 staining patterns between left and right FN appeared identical to that described above for 24 h and 4-day operated animals (not shown). Omitting the primary antibody resulted in negative immunostaining (not shown). DISCUSSION
The present results demonstrate that facial nerve axotomy causes a larger and longer increase of GRP78 in the ipsilateral FN than in the contralateral FN. Axotomy induces GRP78 protein levels in ipsilateral FN in a time-dependent manner, beginning at 12 h and reaching maximal concentrations at 24 h. Although protein concentrations decreased thereafter, they are maintained elevated over sham-operated controls. The immunocytochemical studies done in conjunction with the one-dimensional gel/Western blot experiments showed that GRP78-positive immunostaining was localized primarily in FN motoneurons. The distribution of the epitope was restricted to the cytoplasm, appeared punctate, and is compatible with the described localization of GRP78 within the ER (14). The postaxotomy induction of GRP78 protein levels in right FN observed in the biochemical studies could be also detected at the morphological level, by direct comparison between right (operated) and left (control) FN for each case analyzed. Namely, right FN motoneurons appeared more intensely stained than left FN controls. Further, some elevation in staining of the right FN neuropil appeared, perhaps related to increased dendritic immunoreactivity. Western blot analysis showed that the monoclonal anti-GRP78 antibody recognized another related stress protein, GRP94. It is possible that cross-reactivity to GRP94 contributed, in part, to the neuronal immunostaining with this antibody. However, the polyclonal antibody used recognized only GRP78 (determined by immunoblotting, not shown) and the postaxotomy induction of this protein in right FN motoneurons at 24 h could be confirmed clearly. Further, the intracellular distribution of the protein remained unchanged between early and late time points (i.e., between 6 and 24 h, in which the maximal increase was observed by immunoblotting). The nucleolar immunoreactivity observed at 7 days postaxotomy was apparently not related to treatment, since it was present in both intact
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´ BAL, AND KREUTZBERG MORENO-FLORES, OLAZA
FIG. 4. Cellular localization of GRP78 protein after axotomy by immunohistochemistry (monoclonal antibody). GRP78 immunostaining of left (A, C, E) and right (B, D, F) facial nucleus motoneuron at 24 h (A, B), 4 days (C, D), and 7 days (E, F) postaxotomy of the right facial nerve. Positive immunostaining was localized in the cytoplasm without nuclear staining. Prominent GRP78-immunostaining appeared in right FN motoneurons at 24 h postaxotomy as compared with the contralateral, unoperated control (c.f. A and B). This augmentation was also observed at 4 days (c.f. C and D) and 7 days postaxotomy (c.f. E and F). Bar, 50 µm.
AXOTOMY REGULATION OF GRP78 IN FACIAL NUCLEUS
and injured motoneurons. Last, GRP78 protein levels in left FN were in most cases higher than in right FN, except at 24 h after axotomy (compare Figs. 1A and 1B, pooled results). This tendency was also observed in a second experiment using separate animals, in which intact, unoperated controls were included (not shown). Therefore, lateral differences in the levels of GRP78 in sham-operated animals was not due to the trauma of surgical exposure of the facial nerve. We have also observed some increases in GRP78 in left FN after right facial nerve axotomy, as compared to controls. It remains unclear why this occurs, but it is possible that initial axotomy-induced loss of motor function of the right vibrissae may result in compensatory functional activity of the left vibrissae. Such compensatory increases in left FN could account for the rise in protein levels detected by immunoblotting. In conclusion, axotomy causes a larger and longer increase of GRP78 in the lesioned FN. At present, there is no precise information on the physiological function of GRP78 in the FN. In eukaryotic cells, GRP78 is a member of the stress protein family and presents near 60% homology with another stress protein, heat shock 70 kDa (hsp70) (14). GRP78 responds to glucose starvation (23), a condition which has been shown to inhibit N-linked glycosylation of proteins and is induced by glycosylation inhibitors (18, 20). Following axotomy of the facial nerve, GRP78 levels increased at 12 h, before the maximum glucose utilization described for axotomized facial and hypoglossal nuclei occurs (9, 22, 24) and peaks at 24 h, coinciding with the earliest time point of peak FN glucose metabolism thus far reported. In this context, axotomy may cause early glucose necessities in FN motoneurons, resulting from enhanced necessity for protein synthesis and glycosylation in early stages after the injury. Since GRP78 is an ER protein implicated in protein folding and subunit assembly (2–4, 19) and since increases in protein synthesis occur in hypoglossal motor nucleus following axotomy (24), induction of GRP78 in axotomized FN may occur in parallel to the initial glucose use and cellular activation and be an interrelated event in protein folding and glycosylation in the ER. Thus, GRP78 and the ubiquitous glucose transporter GLUT-1 expressions are regulated similarly by glucose starvation and glycosylation inhibitors (25). Increased metabolic activity after nerve injury might also result in the concurrent accumulation of ER unglycosylated proteins. These abnormal proteins might undergo aberrant folding or form damaging aggregates (see Munro and Pelham (14), for discussion). Both GRPs and hsps are produced in response to increases in aberrant proteins (8, 13) and GRP78 recognizes and binds malfolded or aberrantly glycosylated polypeptides in vitro (7). Furthermore, the promoter region of GRP78 responsive to the accumulation of abnormal
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proteins in ER has been mapped (see Hightower (5), for discussion). It is therefore possible that newly synthesized GRP78 in axotomized FN is a direct consequence of stress and/or the result of regeneration necessities. In related work, Olaza´bal et al. (17) have shown that facial nerve axotomy induces the expression of constitutive hsp70 in axotomized FN. Protein levels peaked at 3 days postaxotomy and remained elevated thereafter. It is of interest that the pattern of GRP78 induction is similar to that of hsp70. Moreover, preliminary experiments using a cDNA probe complementary to GRP78 (kind gift of Dr. Hugh Pelham) have thus far shown a biphasic postaxotomy induction of GRP78 mRNA in operated FN, occurring as early as 1 h and rising again at 6 and 12 h (not shown). In agreement with these data, Lowenstein et al. (12) have described rapid, early increases in GRP78, GRP94, and hsp72 gene expression in different models of acute CNS trauma. The return to baseline for the GRPs tended to exceed 24 h. Taken together, these results lend support for a role of GRP78 in the mechanisms underlying motoneuron regeneration. The functional significance of these protein families in the response of the nervous system to injury and in peripheral nerve regeneration deserve further analysis. ACKNOWLEDGMENTS We thank Kerstin Wulf for her technical assistance and Dr. F. Wandosell and Dr. F. Lim for invaluable help and discussion in the preparation of the manuscript.
REFERENCES 1.
2.
3. 4.
5. 6.
7.
8.
Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248–254. Gething, M.-J., K. McCammon, and J. Sambrook. 1986. Expression of wild type and mutant forms of influenza hemagglutin: The role of folding in intracellular transport. Cell 46: 939–950. Haas, I. G., and M. Wabi. 1983. Immunoglobulin heavy chain binding protein. Nature 306: 387–389. Hendershot, L., D. Bole, and J. F. Kearney. 1987. The role of immunoglobulin heavy chain binding protein in immunoglobulin transport. Immunol. Today 8: 111–114. Hightower, L. E. 1991. Heat shock, stress proteins, chaperones, and proteotoxicity. Cell 66: 191–197. Hightower, L. E., and F. P. White. 1981. Cellular responses to stress: Comparison of a family of 71–73 kilodalton proteins rapidly synthesized in rat tissue slices and canavanine treated cells in culture. J. Cell. Physiol. 108: 261–275. Kassenbrock, C. K., P. D. Garcia, P. Walter, and R. B. Kelly. 1988. Heavy-chain binding protein recognizes aberrant polypeptides translocated in vitro. Nature 333: 90–93. Kozutsumi, Y., M. Segal, K. Normington, M.-J. Gething, and J. Sambrook. 1988. The presence of malfolded proteins in the endoplasmic reticulum signals the induction of glucose-regulated proteins. Nature 332: 462–464.
16
´ BAL, AND KREUTZBERG MORENO-FLORES, OLAZA
9.
Kreutzberg, G. W., and H. Emmert. 1980. Glucose utilization of motor nuclei during regeneration: A [14C]2-deoxyglucose study. Exp. Neurol. 70: 712–716.
10.
Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685.
11.
Lieberman, A. R. 1971. The axon reaction: A review of the principal features of perikaryal responses to axon injury. Int. Rev. Neurobiol. 14: 50–124.
12.
Lowenstein, D. H., R. P. Gwinn, M. S. Seren, R. P. Simon, and T. K. McIntosh. 1994. Increased expression of mRNA encoding calbinding-D28K, the glucose-regulated proteins, or the 72 kDA heat-shock protein in three models of acute CNS injury. Mol. Brain Res. 22: 299–308.
13.
Munro, S., and H. R. B. Pelham. 1985. What turns on heat shock genes? Nature 317: 477–478.
14.
Munro, S., and H. R. B. Pelham. 1986. An Hsp70-like protein in the ER: Identity with the 78 kd glucose-regulated protein and immunoglobulin heavy chain binding protein. Cell 46: 291–300.
15.
Munro, S., and H. R. B. Pelham. 1987. A C-terminal signal prevents secretion of luminal ER proteins. Cell 48: 899–907.
Murray, M. 1973. 3H-Uridine incorporation by regenerating retinal ganglion cells of goldfish. Exp. Neurol. 39: 489–497. 17. Olaza´bal, U. E., M. Reddington, and G. W. Kreutzberg. 1993. Expression and regulation of heat shock proteins following facial nerve axotomy in rats. Soc. Neurosci. Abs. 19: 1313. 18. Olden, K., R. M. Pratt, C. Jaworski, and K. M. Yamada. 1979. Evidence for role of glycoprotein carbohydrates in membrane 16.
19.
20.
21.
22.
23.
24.
25.
transport: Specific inhibition by tunicamycin. Proc. Natl. Acad. Sci. USA 76: 791–795. Pelham, H. R. B. 1986. Speculations on the functions of the major heat shock and glucose-regulated proteins. Cell 46: 959–961. Pouysse´gur, J., R. P. C. Shiu, and I. Pastan. 1977. Induction of two transformation-sensitive membrane polypeptides in normal fibroblasts by a block in glycoprotein synthesis or glucose deprivation. Cell 11: 941–947. Resendez, E., J. W. Attenello, A. Grafsky, C. S. Chang, and A. S. Lee. 1985. Calcium ionophore A23187 induces expression of glucose-regulated genes and their heterologous fusion genes. Mol. Cell. Biol. 5: 1212–1219. Singer, P., and S. Mehler. 1980. 2-Deoxy[14C]glucose uptake in the rat hypoglossal nucleus after nerve transection. Exp. Neurol. 69: 617–626. Shiu, R. P. C., J. Pouysse´gur, and I. Pastan. 1977. Glucose depletion accounts for the induction of two transformationsensitive membrane proteins in Rous sarcoma virus-transformed chick embryo fibroblasts. Proc. Natl. Acad. Sci. USA 74: 3840–3844. Smith, C. B., A. M. Crane, M. Kadekaro, B. W. Agranoff, and L. Sokoloff. 1984. Stimulation of protein synthesis and glucose utilization in the hypoglossal nucleus induced by axotomy. J. Neuroimmunol. 10: 2489–2496. Wertheimer, E., S. Sasson, and Y. Ben-Neriah. 1991. The ubiquitous glucose transporter GLUT-1 belongs to the glucoseregulated protein family of stress-inducible proteins. Proc. Natl. Acad. Sci. USA 88: 2525–2529.
146, 10–16 (1997)
EN976526
Axotomy Increases the Expression of Glucose-Regulated Protein 78 kDa in Rat Facial Nucleus M. Teresa Moreno-Flores,1 Ursula E. Olaza´bal, and Georg W. Kreutzberg2 Max-Planck-Institute of Psychiatry, Department of Neuromorphology, Am Klopferspitz 18a, 82152 Martinsried, Germany
supplying energy during axonal reaction and regeneration have been described (9, 16, 22, 24). In facial and hypoglossal motor nuclei, there is an increase in glucose utilization and protein synthesis after axotomy of their respective nerves (9, 22, 24), which was found as early as 24 h postaxotomy and persisted after the functional recovery. A variety of stressful conditions are known to induce the expression of heat shock proteins (hsp) (6). Stress proteins include the glucose-regulated protein (GRP) family. GRPs share high homology with some hsps, are localized in endoplasmic reticulum (ER) (5, 14, 15), are implicated in cellular protein folding and subunit assembly, and are induced under conditions of glucose starvation (2–4, 19, 23). In addition, GRPs are induced by amino acid analogs, as are hsps, and by GRP-selective stressors, such as glycosylation inhibitors and Ca21 ionophores (6, 18, 20, 21). Although information is available on early postaxotomy glucose utilization and protein synthesis, the cellular mechanisms underlying these responses are not clearly understood. The present studies were, therefore, carried out to determine the effect of axotomy on the expression and regulation of GRP 78 kDa (GRP78) in the facial nucleus (FN).
Nerve injuries lead to metabolic and morphological changes in the cell bodies of the neurons of origin. Increases in glucose turnover in axotomized facial and hypoglossal motor nuclei have been described. Glucoseregulated protein 78 kDa (GRP78) is implicated in cellular protein folding and subunit assembly and responds to glucose deficiency. We performed Western blot and immunohistochemistry to determine the effect of axotomy on the expression and regulation of GRP78 in the facial nucleus (FN). Facial nerve axotomy caused a larger and longer increase of GRP78 in the ipsilateral FN than in the contralateral FN. In right ipsilateral FN, axotomy resulted in elevation of GRP78 protein levels, first detected at 12 h and which reached significant, maximal induction at 24 h (75 6 27% increase). GRP78 protein levels decreased at later time points, but remained elevated over sham-operated controls. In contrast, no significant increase in GRP78 concentrations was found in contralateral left FN. Immunocytochemically, positive GRP78 staining was found mainly in the cytoplasm of motoneurons; there was no nuclear staining. Prominent GRP78-immunostaining appeared in axotomized motoneurons at 24 h postaxotomy as compared with the contralateral, unoperated controls. This augmentation was also observed at 4 and 7 days postaxotomy. The possibility that glucose metabolism and GRP78 levels are two parallel events in the injured facial nucleus is discussed. r 1997
MATERIALS AND METHODS
Animals and Surgery
Academic Press
A total of 63 Wistar male rats (250–300 g) were used in biochemical and morphological studies. The animals were housed in small groups on a 12:12 light:dark cycle and provided with food and water ad libitum. Axotomy time course studies were done by transecting the right facial nerve at the stylomastoid foramen under ether anesthesia. For biochemical studies, animals were sacrificed at 6 h (n 5 4), 12 h (n 5 10), 24 h (n 5 8), 72 h (3 days; n 5 10), 168 h (7 days; n 5 6), or 336 h (14 days; n 5 6) following axotomy by brief exposure to CO2 (30 s). The facial nuclei (FN) (right, operated, ipsilateral to the transection; left, unoperated, contralateral to the transection) were microdissected from a 2-mm slice and stored at 270°C. Three rats were used as shamoperated controls (S/O; exposing facial nerve without
INTRODUCTION
Axotomy and other nerve injuries lead to metabolic and morphological changes in the cell bodies of the neurons of origin (11). In the peripheral nervous system, nerve regeneration and functional restoration usually follow these processes. Metabolic changes in RNA, protein synthesis, and in oxidative metabolism
1 Present address: Centro de Biologı´a Molecular ‘‘Severo Ochoa’’ (CSIC-UAM), Facultad de Ciencias, Universidad Auto´noma, 28049 Madrid, Spain. Fax: (00341) 397 48 70. 2 Reprint requests to Georg W. Kreutzberg.
0014-4886/97 $25.00 Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.
10
AXOTOMY REGULATION OF GRP78 IN FACIAL NUCLEUS
transection; survival time 24 h) and two additional rats were used as unoperated ‘‘intact’’ controls. In morphological studies, animals were operated as described and sacrificed at 6 h (n 5 2), 24 h (n 5 5), 4 days (n 5 3), 7 days (n 5 2), or 15 days (n 5 2) later by trans-cardial perfusion with isotonic saline solution (0.9% NaCl) for 10 min under deep chloral hydrate anesthesia (30% solution, 50 mg/kg body weight). The brains were removed, immediately frozen, and stored at 270°C until ready to use. Materials Immunological studies were done using two different antibodies directed against GRP78 protein: (i) A mouse monoclonal anti-GRP78 antibody (StressGen, B.C., Canada), prepared against an heptapeptide from the carboxy terminus of rat GRP78. The antibody identified primarily GRP78, but also crossreacted with another glucose stress protein, GRP 94 kDa. (ii) A rabbit polyclonal anti-GRP78 (StressGen), prepared against a synthetic peptide from the carboxy terminus of human GRP78. This antibody did not show cross-reactivity with other proteins. Pure hsp70 protein was also obtained from StressGen. Polyvinyldifluoride membranes were obtained from Schleicher & Schuell. For immunoblotting, secondary antibodies and enhanced chemiluminescence kits were obtained from Amersham. Secondary antibodies used in immunocytochemical studies, as well as normal sera and avidin–biotin Vectastain kits, were purchased from Vector Labs. Biochemical Studies Electrophoresis. Extracted facial nuclei were prepared in sodium dodecyl sulfate (SDS) sample buffer, containing 0.5 M Tris base, 0.1 M EDTA, 2% SDS, 10% glycerol, and 10% mercaptoethanol and heat denatured. Samples were normalized with respect to total protein concentration (1) and 10 µg protein from each were resolved on two-phase one-dimensional gels consisting of a 4% acrylamide upper stacking gel and a 10% acrylamide lower resolving gel (6 3 9 cm) (10), using a Hoeffer SE 215 mini gel apparatus. On each gel, single samples from the entire axotomy time course (S/O, 6 h, 12 h, 24 h, 3 days, 7 days, and 14 days) for right (operated) or left (control) nuclei were resolved. Thus, every gel represented an individual time course analysis for right and/or left FN. A total of 19 gels were electrophoresed at constant voltage (150 V) and time (1:15 h), which comprised two independent experiments. Western blots/enhanced chemiluminescence. Proteins were captured on polyvinyldifluoride filters. Immunoblots were blocked in Tween buffer (0.01 M Tris base, 0.5 M NaCl, 0.5% Tween 20) containing 10% nonfat dry milk and incubated in the monoclonal anti-GRP78 antibody (1:500). A second blocking step in 5% normal
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serum was done, followed by incubation in the horseradish peroxidase-conjugated secondary antibody. Immunoblots were washed overnight in Tween buffer prior to developing with enhanced chemiluminescence reagent. Specific GRP78 bands were detected on film using enhanced chemiluminescence and exposing to film at various times to optimize signal visualization. These parameters were then maintained constant for all blots thereafter. Specific bands were digitized and analyzed using a laser scanner equipped with Image Quant analysis software (Molecular Dynamics, CA). Once bands were defined, optical densities (OD) units of signal over background were recorded and averaged across blots. Results are expressed as mean OD units (GRP78 immunoreactivity) 6 standard error (SEM) for every experimental condition. Further, the mean and coefficient of variance were calculated for every experimental condition and compared to controls. If the two mean values differed more than twice the coefficient of variance, these values were additionally compared using an unpaired two-tailed student’s t test. Immunohistochemical Studies The cellular localization of GRP78 protein was examined by immunohistochemistry, to determine the cell types in FN which could be involved in the response of this protein to axotomy. Briefly, fresh 20-µm cryosections containing the facial nuclei were incubated in a 1:200 dilution of the primary anti-GRP78 antibody (monoclonal or polyclonal) followed by incubation in the biotinylated secondary antibody (for each corresponding primary antibody). Sections were immunostained using the Vectastain ABC kit (Vector Lab.) and developed in diaminobenzidine/H2O2, dehydrated, coverslipped, and examined using light microscopy. Control Conditions Since GRP78 and hsp70 share approximately 60% homology (14, 15), it was important to determine the specificity of the GRP78 antibody. Preadsorption control experiments were carried out on alternate sections from 1- and 4-day postaxotomy animals by first incubating pure hsp70 protein with the primary anti-grp78 antibody and then applying to tissue sections. Additional controls were also involved omitting the primary GRP78 antibody from the immunostaining procedure. RESULTS
Effects of Axotomy on GRP78 Protein Levels: Western Blots Intact and sham-operated animals were compared to establish control levels of GRP78 in FN. We found differences in the baseline for right and left FN, both in intact and sham-operated animals. For control intact
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´ BAL, AND KREUTZBERG MORENO-FLORES, OLAZA
animals, the left side had a mean of 171% with respect to the right FN that represented 100% in values of optical density units. For control sham-operated animals the mean was 130% for the left FN, contralateral to the facial nerve exposed in the surgery without transection, with respect to 100% values of right/
FIG. 2. Percentage of control increase in GRP78 immunoreactivity in right (axotomized; filled boxes) and left (unoperated; unfilled boxes) facial nucleus at different times postaxotomy, in respect to their controls. In single immunoblots (right or left), percentage increases over controls were determined for each experimental condition and pooled across blots from two independent experiments. Results are expressed as mean percentage increases 6 standard error.
FIG. 1. (A) Time course induction of GRP78 protein in the right facial nucleus in response to axotomy. Sample preparation and Western blot analysis using anti-grp78 monoclonal antibody was done as described under Materials and Methods. 0 hours represents the sham-operated control rats in which the right facial nerve was exposed but not transected. At each time point, the results are expressed as mean optical density units 6 standard error. Right FN axotomy caused a significant rise in GRP78 concentrations, which was maximal at 24 h (P 5 0.0007, unpaired two-tailed t test). (B) GRP78 immunoreactivity in the left facial nucleus after axotomy of the contralateral facial nerve. Time course studies were carried out as for right FN and the results are expressed as mean optical density units 6 standard error for every experimental condition. Following right FN transection, an increase in GRP78 levels were observed at 12 h, but did not significantly differ from sham-op controls (P 5 0.22; unpaired two-tailed t test). Concentrations decreased at 24 h and then returned to baseline. FN, facial nucleus; GRP78, glucoseregulated protein 78 kDa.
ipsilateral FN. Therefore the differences in the baseline for both FN were not due to surgery stress. Means 6 SEM at each time point for right or left FN are shown in Fig. 1, representative of two separate independent experiments. In right FN, axotomy resulted in an elevation of GRP78 protein levels, first detected at 12 h and which reached maximal induction at 24 h (P 5 0.0007; unpaired, two-tailed t test; Fig. 1A). This induction, at 24 h, represented an 75 6 27% increase and was consistent in 7 of 7 rats evaluated, as shown in Fig. 2. These data are the pooled values of two independent experiments. Due to the uniformity in the GRP78 response in operated FN for all animals studied at this time point, the standard error of the mean value at this time point was consistently small across independent experiments. Similarly, the variation observed for S/O right FN was also small since no change in GRP78 levels were found between animals. GRP78 protein levels decreased at 3 days postaxotomy and at later time points, but remained elevated over S/O controls. The corresponding variations were also higher. In contrast, in the corresponding contralateral FN, maximal levels were observed at 12 h postaxotomy (Figs. 1B and 2; 39 6 11%; P 5 0.22; n 5 9), which decreased at 24 h (19 6 10%; n 5 7; Fig. 2) and returned to baseline levels thereafter. Notably, the response at 12 h in the unoperated FN was less pronounced that in the operated, right nucleus (Fig. 2; 39 6 11% vs 65 6 21%), was not significant and was transient, in comparison with the operated side in which GRP78 response peaked at 24 h and maintained higher than the baseline for 14 days. Therefore, GRP78 concentrations in left FN varied in response to axotomy of the contralateral facial nerve, but insignificantly.
AXOTOMY REGULATION OF GRP78 IN FACIAL NUCLEUS
FIG. 3. Single Western blot analysis of axotomy time course effects on GRP78 protein in (A) right facial nucleus and (B) left facial nucleus. (A) In right axotomized facial nucleus, GRP78-specific band intensity is greater at 12 h versus control (c.f. lanes 1 and 3), peaks at 24 h (lane 4), and begins to decrease at 72 h, yet remaining higher than control (c.f. lanes 1, 5, 6, and 7). (B) GRP78 band immunoreactivity in left, unoperated facial nucleus transiently increased at 12 h after axotomy of the right facial nerve (lane 3) and returned to baseline thereafter.
Individual Western blot experiments from right (A, axotomized) and left (B, unoperated) FN are shown in Fig. 3. These are representative cases of the pooled data for each analysis. Immunoreactive bands represent tissue sampled from independent animals and processed as described under Materials and Methods. In single immunoblots, intensely reactive bands at 78 kDa were observed in all lanes. In right FN (Fig. 3A, top), postaxotomy GRP78 increases were first detectable at 12 h (lane 3). Immunoreactive band intensity was maximal at 24 h (lane 4), being markedly higher than in S/O controls or 6 h postaxotomy (c.f. lanes 1, 2, and 4). GRP78 protein concentrations decreased at 3 days, but were nevertheless elevated over the postaxotomy time course tested (lanes 5, 6, and 7). In single, left FN immunoblots (Fig. 3B, bottom), a transient rise in GRP78 protein was noted at 12 h after axotomy of the contralateral nerve (c.f. lanes 1 and 3), which later returned to control levels (lanes 4–7). GRP78 Immunohistochemistry Both monoclonal and polyclonal antibodies presented similar patterns of immunostaining across all animals under the conditions tested. In all sections, these antibodies stained primarily neurons and appeared as either diffuse or punctate in the cytoplasmic space; there was no nuclear staining (Fig. 4). In FN, GRP78immunoreactivity appeared in motoneurons. Shortly following axotomy (6 h), no detectable changes in immunostaining of motoneurons were found (not shown). However, prominent GRP78-immunostaining of right FN motoneurons appeared at 24 h postaxotomy (Fig. 4B), as compared with the contralateral, unoperated FN (Fig. 4A). This augmentation was detected in all animals studied at this time point and was consis-
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tent with that observed in the Western blot analysis (Fig. 1). A slight increase in staining intensity of the neuropil in operated FN could be also seen. Similar increases in motoneuron immunostaining were also observed at 4 days (Fig. 4C, left FN, and 4D, right FN) and 7 days (Fig. 4E, left FN, and 4F, right FN) after transection, except that higher individual staining variability was noted and thus resulting in less marked differences in right–left FN comparisons. These findings were also noted at the level of immunoblotting (Fig. 1). In preadsorption control experiments, positive GRP78 staining patterns between left and right FN appeared identical to that described above for 24 h and 4-day operated animals (not shown). Omitting the primary antibody resulted in negative immunostaining (not shown). DISCUSSION
The present results demonstrate that facial nerve axotomy causes a larger and longer increase of GRP78 in the ipsilateral FN than in the contralateral FN. Axotomy induces GRP78 protein levels in ipsilateral FN in a time-dependent manner, beginning at 12 h and reaching maximal concentrations at 24 h. Although protein concentrations decreased thereafter, they are maintained elevated over sham-operated controls. The immunocytochemical studies done in conjunction with the one-dimensional gel/Western blot experiments showed that GRP78-positive immunostaining was localized primarily in FN motoneurons. The distribution of the epitope was restricted to the cytoplasm, appeared punctate, and is compatible with the described localization of GRP78 within the ER (14). The postaxotomy induction of GRP78 protein levels in right FN observed in the biochemical studies could be also detected at the morphological level, by direct comparison between right (operated) and left (control) FN for each case analyzed. Namely, right FN motoneurons appeared more intensely stained than left FN controls. Further, some elevation in staining of the right FN neuropil appeared, perhaps related to increased dendritic immunoreactivity. Western blot analysis showed that the monoclonal anti-GRP78 antibody recognized another related stress protein, GRP94. It is possible that cross-reactivity to GRP94 contributed, in part, to the neuronal immunostaining with this antibody. However, the polyclonal antibody used recognized only GRP78 (determined by immunoblotting, not shown) and the postaxotomy induction of this protein in right FN motoneurons at 24 h could be confirmed clearly. Further, the intracellular distribution of the protein remained unchanged between early and late time points (i.e., between 6 and 24 h, in which the maximal increase was observed by immunoblotting). The nucleolar immunoreactivity observed at 7 days postaxotomy was apparently not related to treatment, since it was present in both intact
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´ BAL, AND KREUTZBERG MORENO-FLORES, OLAZA
FIG. 4. Cellular localization of GRP78 protein after axotomy by immunohistochemistry (monoclonal antibody). GRP78 immunostaining of left (A, C, E) and right (B, D, F) facial nucleus motoneuron at 24 h (A, B), 4 days (C, D), and 7 days (E, F) postaxotomy of the right facial nerve. Positive immunostaining was localized in the cytoplasm without nuclear staining. Prominent GRP78-immunostaining appeared in right FN motoneurons at 24 h postaxotomy as compared with the contralateral, unoperated control (c.f. A and B). This augmentation was also observed at 4 days (c.f. C and D) and 7 days postaxotomy (c.f. E and F). Bar, 50 µm.
AXOTOMY REGULATION OF GRP78 IN FACIAL NUCLEUS
and injured motoneurons. Last, GRP78 protein levels in left FN were in most cases higher than in right FN, except at 24 h after axotomy (compare Figs. 1A and 1B, pooled results). This tendency was also observed in a second experiment using separate animals, in which intact, unoperated controls were included (not shown). Therefore, lateral differences in the levels of GRP78 in sham-operated animals was not due to the trauma of surgical exposure of the facial nerve. We have also observed some increases in GRP78 in left FN after right facial nerve axotomy, as compared to controls. It remains unclear why this occurs, but it is possible that initial axotomy-induced loss of motor function of the right vibrissae may result in compensatory functional activity of the left vibrissae. Such compensatory increases in left FN could account for the rise in protein levels detected by immunoblotting. In conclusion, axotomy causes a larger and longer increase of GRP78 in the lesioned FN. At present, there is no precise information on the physiological function of GRP78 in the FN. In eukaryotic cells, GRP78 is a member of the stress protein family and presents near 60% homology with another stress protein, heat shock 70 kDa (hsp70) (14). GRP78 responds to glucose starvation (23), a condition which has been shown to inhibit N-linked glycosylation of proteins and is induced by glycosylation inhibitors (18, 20). Following axotomy of the facial nerve, GRP78 levels increased at 12 h, before the maximum glucose utilization described for axotomized facial and hypoglossal nuclei occurs (9, 22, 24) and peaks at 24 h, coinciding with the earliest time point of peak FN glucose metabolism thus far reported. In this context, axotomy may cause early glucose necessities in FN motoneurons, resulting from enhanced necessity for protein synthesis and glycosylation in early stages after the injury. Since GRP78 is an ER protein implicated in protein folding and subunit assembly (2–4, 19) and since increases in protein synthesis occur in hypoglossal motor nucleus following axotomy (24), induction of GRP78 in axotomized FN may occur in parallel to the initial glucose use and cellular activation and be an interrelated event in protein folding and glycosylation in the ER. Thus, GRP78 and the ubiquitous glucose transporter GLUT-1 expressions are regulated similarly by glucose starvation and glycosylation inhibitors (25). Increased metabolic activity after nerve injury might also result in the concurrent accumulation of ER unglycosylated proteins. These abnormal proteins might undergo aberrant folding or form damaging aggregates (see Munro and Pelham (14), for discussion). Both GRPs and hsps are produced in response to increases in aberrant proteins (8, 13) and GRP78 recognizes and binds malfolded or aberrantly glycosylated polypeptides in vitro (7). Furthermore, the promoter region of GRP78 responsive to the accumulation of abnormal
15
proteins in ER has been mapped (see Hightower (5), for discussion). It is therefore possible that newly synthesized GRP78 in axotomized FN is a direct consequence of stress and/or the result of regeneration necessities. In related work, Olaza´bal et al. (17) have shown that facial nerve axotomy induces the expression of constitutive hsp70 in axotomized FN. Protein levels peaked at 3 days postaxotomy and remained elevated thereafter. It is of interest that the pattern of GRP78 induction is similar to that of hsp70. Moreover, preliminary experiments using a cDNA probe complementary to GRP78 (kind gift of Dr. Hugh Pelham) have thus far shown a biphasic postaxotomy induction of GRP78 mRNA in operated FN, occurring as early as 1 h and rising again at 6 and 12 h (not shown). In agreement with these data, Lowenstein et al. (12) have described rapid, early increases in GRP78, GRP94, and hsp72 gene expression in different models of acute CNS trauma. The return to baseline for the GRPs tended to exceed 24 h. Taken together, these results lend support for a role of GRP78 in the mechanisms underlying motoneuron regeneration. The functional significance of these protein families in the response of the nervous system to injury and in peripheral nerve regeneration deserve further analysis. ACKNOWLEDGMENTS We thank Kerstin Wulf for her technical assistance and Dr. F. Wandosell and Dr. F. Lim for invaluable help and discussion in the preparation of the manuscript.
REFERENCES 1.
2.
3. 4.
5. 6.
7.
8.
Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248–254. Gething, M.-J., K. McCammon, and J. Sambrook. 1986. Expression of wild type and mutant forms of influenza hemagglutin: The role of folding in intracellular transport. Cell 46: 939–950. Haas, I. G., and M. Wabi. 1983. Immunoglobulin heavy chain binding protein. Nature 306: 387–389. Hendershot, L., D. Bole, and J. F. Kearney. 1987. The role of immunoglobulin heavy chain binding protein in immunoglobulin transport. Immunol. Today 8: 111–114. Hightower, L. E. 1991. Heat shock, stress proteins, chaperones, and proteotoxicity. Cell 66: 191–197. Hightower, L. E., and F. P. White. 1981. Cellular responses to stress: Comparison of a family of 71–73 kilodalton proteins rapidly synthesized in rat tissue slices and canavanine treated cells in culture. J. Cell. Physiol. 108: 261–275. Kassenbrock, C. K., P. D. Garcia, P. Walter, and R. B. Kelly. 1988. Heavy-chain binding protein recognizes aberrant polypeptides translocated in vitro. Nature 333: 90–93. Kozutsumi, Y., M. Segal, K. Normington, M.-J. Gething, and J. Sambrook. 1988. The presence of malfolded proteins in the endoplasmic reticulum signals the induction of glucose-regulated proteins. Nature 332: 462–464.
16
´ BAL, AND KREUTZBERG MORENO-FLORES, OLAZA
9.
Kreutzberg, G. W., and H. Emmert. 1980. Glucose utilization of motor nuclei during regeneration: A [14C]2-deoxyglucose study. Exp. Neurol. 70: 712–716.
10.
Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685.
11.
Lieberman, A. R. 1971. The axon reaction: A review of the principal features of perikaryal responses to axon injury. Int. Rev. Neurobiol. 14: 50–124.
12.
Lowenstein, D. H., R. P. Gwinn, M. S. Seren, R. P. Simon, and T. K. McIntosh. 1994. Increased expression of mRNA encoding calbinding-D28K, the glucose-regulated proteins, or the 72 kDA heat-shock protein in three models of acute CNS injury. Mol. Brain Res. 22: 299–308.
13.
Munro, S., and H. R. B. Pelham. 1985. What turns on heat shock genes? Nature 317: 477–478.
14.
Munro, S., and H. R. B. Pelham. 1986. An Hsp70-like protein in the ER: Identity with the 78 kd glucose-regulated protein and immunoglobulin heavy chain binding protein. Cell 46: 291–300.
15.
Munro, S., and H. R. B. Pelham. 1987. A C-terminal signal prevents secretion of luminal ER proteins. Cell 48: 899–907.
Murray, M. 1973. 3H-Uridine incorporation by regenerating retinal ganglion cells of goldfish. Exp. Neurol. 39: 489–497. 17. Olaza´bal, U. E., M. Reddington, and G. W. Kreutzberg. 1993. Expression and regulation of heat shock proteins following facial nerve axotomy in rats. Soc. Neurosci. Abs. 19: 1313. 18. Olden, K., R. M. Pratt, C. Jaworski, and K. M. Yamada. 1979. Evidence for role of glycoprotein carbohydrates in membrane 16.
19.
20.
21.
22.
23.
24.
25.
transport: Specific inhibition by tunicamycin. Proc. Natl. Acad. Sci. USA 76: 791–795. Pelham, H. R. B. 1986. Speculations on the functions of the major heat shock and glucose-regulated proteins. Cell 46: 959–961. Pouysse´gur, J., R. P. C. Shiu, and I. Pastan. 1977. Induction of two transformation-sensitive membrane polypeptides in normal fibroblasts by a block in glycoprotein synthesis or glucose deprivation. Cell 11: 941–947. Resendez, E., J. W. Attenello, A. Grafsky, C. S. Chang, and A. S. Lee. 1985. Calcium ionophore A23187 induces expression of glucose-regulated genes and their heterologous fusion genes. Mol. Cell. Biol. 5: 1212–1219. Singer, P., and S. Mehler. 1980. 2-Deoxy[14C]glucose uptake in the rat hypoglossal nucleus after nerve transection. Exp. Neurol. 69: 617–626. Shiu, R. P. C., J. Pouysse´gur, and I. Pastan. 1977. Glucose depletion accounts for the induction of two transformationsensitive membrane proteins in Rous sarcoma virus-transformed chick embryo fibroblasts. Proc. Natl. Acad. Sci. USA 74: 3840–3844. Smith, C. B., A. M. Crane, M. Kadekaro, B. W. Agranoff, and L. Sokoloff. 1984. Stimulation of protein synthesis and glucose utilization in the hypoglossal nucleus induced by axotomy. J. Neuroimmunol. 10: 2489–2496. Wertheimer, E., S. Sasson, and Y. Ben-Neriah. 1991. The ubiquitous glucose transporter GLUT-1 belongs to the glucoseregulated protein family of stress-inducible proteins. Proc. Natl. Acad. Sci. USA 88: 2525–2529.