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Opposite effects of axon damage on heat shock proteins (hsp 70) and ubiquitin contents in motor neurons of neuropathic rats
Neuroscience Letters, 153 (1993) 49-52 Elsevier Scientific Publishers Ireland Ltd.
49
NSL 09422
Opposite effects of axon damage on heat shock proteins (hsp 70) and ubiquitin contents in motor neurons of neuropathic rats M. Lant6ri-Minet b, J.A. Desmeules c and D. Men6trey a aInstitut National de la Santd et de la Recherche Mddicale Unitd 161, Paris (France), bLaboratoire de Neuro-oncologie et Neurosciences Cliniques, Service de Neurologie du Centre Hospitalo-Universitaire de Nice, Nice (France) and CDivision de Pharmacologie Clinique, Hopital Cantonal Universitaire de Genkve,. GenOve (Switzerland) (Received 29 September 1992; Revised version received 13 December 1992; Accepted 23 December 1992 Key words: Nerve ligature; Chaperon; Proteolytic factor; c-JUN; Nerve growth factor receptor; Calcitonin gene-related peptide; Motoneuron; Immunocytochemistry Alteration in the motoneurone contents of heat shock protein (hsp 70) and ubiquitin were studied in rats which had been subject to loose ligation of one common sciatic nerve. This results in a unilateral peripheral neuropathy which peaks at 14 days following ligation and is characterized by transient degeneration of both myelinated and unmyelinated nerve fibres, abnormal motor behaviours (posture of the hind limb, walking patterns) and thermal and mechanical allodynia of the hind paw. Hsp 70 and ubiquitin are proteins involved in protein metabolism and their expression is regulated during cellular stress. The contralateral unlesioned side was used as control. Motoneurone staining for hsp 70 and ubiquitin were differentially altered at the peak of the neuropathy. Axon damage resulted in a decrease in hsp 70 labeling while ubiquitin staining increased. At the same time motoneurones undergoing axon damage overstained for the immediate early gene encoded protein c-JUN and for nerve growth factor receptor (rNGF). In contrast, no clear alteration was seen, at that time, in the intensity of labeling for calcitonin gene-related peptide (CGRP). This study demonstrates that peripheral neuropathy resulting from loose ligation of the common sciatic nerve not only produces sensory alterations as previously reported but also leads to pronounced alterations in motoneurone functioning that could partly explain the observed abnormal motor behaviours. Results are discussed in accordance with presumed roles for hsp 70 and ubiquitin in protein metabolism and in relationship with possible interaction with c-JUN and rNGF expressions.
Protein metabolism in vivo depends on intracellular proteins that are present either constitutively or expressed during physiological stress (see refs. in 5, 17 and 30). Among these proteins are the 70-kDa heat shock proteins (hsp 70) and ubiquitin which act as molecular chaperones or proteolytic factors, respectively. Expression of both constitutive and inducible hsp 70 proteins have been recently reported in the spinal cord [24]; constitutive proteins exist in grey matter areas including motor neurones while hyperthermia-inducible proteins primarily exist in white matter. Ubiquitin has been reported in motoneurones in cases of degenerative diseases [25, 33]. These observations suggest that both hsp 70 and ubiquitin are involved in the turn-over of proteins in motor cells, and that they could be useful markers in demonstrating functional alterations in motoneurone metabolism. Immunoreactivity for hsp 70 and ubiquitin
Correspondence: D. Men6trey, INSERM U 161, 2 rue d'Al6sia, 75014 Paris, France.
was consequently studied in order to evaluate possible motoneuronal alterations consecutive to unilateral peripheral neuropathy resulting from loose ligation of the common sciatic nerve [4]. This disorder which is characterized by transient degeneration of both myelinated and unmyelinated nerve fibers [3, 4, 11], abnormal motor behaviours (posture of the hind limb, walking patterns) and thermal and mechanical allodynia of the hind paw [2, 21] has become a very popular model for chronic pain studies of deafferentation. However, it has never been fully understood if the guarding positions and/or abnormal gait this disorder induces only reflect painful sensations, or are directly related to paresia as a result of the motor deficit. The immunohistochemical study of hsp 70 and ubiquitin contents of motoneurones may provide an easy way to evaluate the degree of the motoneurone impairment. Since motoneurone overexpression of the immediate early gene c-JUN [14, 18, 23], of calcitonin gene-related peptide (CGRP) [1, 12, 28, 29, 31], and of nerve growth factor receptor (rNGF) [9, 20, 32] has been previously noted following axon damage, possible alterations
50 in staining for all of these proteins were also studied in parallel. Ten adult male albino rats of the Sprague-Dawley strain were used. Unilateral ligations of the common sciatic nerve were performed under sodium pentobarbital anaesthesia (50 mg/kg i.p.) by four loose ligatures (5.0 chromic gut) spaced at 1-2 mm intervals. Ligatures are tied loosely to reduce so as not to interrupt blood circulation through the epineural vasculature. Sham procedures which consisted of similar surgery except for ligations were performed on the opposite side. Animals were kept unanaesthetized for the following survival period and managed in accordance with the guidelines of the ethical standards of the IASP committee [8]. Immunohistochemical studies were performed at the peak of the neuropathy (2 weeks). Animals were anaesthetized with sodium pentobarbital and perfused intracardially with 200 ml of 0.1 M phosphate-buffered saline (PBS), pH 7.4, followed by 400 ml of 1% paraformaldehyde in PBS. Tissue was then removed, immersed in 4% fixative solution for 3 h and cryoprotected overnight in phosphate-buffered 30% sucrose solution at 4°C. Frozen serial coronal sections (40 pm) were cut and collected in PBS to be processed as free-floating sections. Sections were incubated in normal serum (10% in phosphate-buffered saline and 0.3% Triton X-100 (PBST)) for 30 min followed by the primary antisera at 4°C overnight. The antisera to hsp 70 and r N G F were monoclonal; hsp 70 antiserum was purchased from Amersham (RPN 1197; Amersham, UK) and run at 1/10,000; r N G F antiserum (Igg 192) was a kind gift of Dr. E.M. Johnson (Washington University, St. Louis, USA) and run at 4/lg/ml. The r N G F antiserum is directed against the low-affinity form of the receptor [6]. The antisera to c-JUN, ubiquitin and C G R P were polyclonal; ubiquitin antiserum was purchased from Chemicon (AB 1690; Temecula, CA, USA) and run at 1/2000; C G R P antiserum was purchased from Cambridge Research Biochemicals (CA 08 220; Northwich, UK) and run at 1/10,000; c-JUN antiserum was a gift of Dr. R. Bravo (Bristol-Meyers Squidd, Princeton, USA) and run at 1/20,000. After incubation in primary antisera, sections were washed in three changes of normal goat serum (1% in PBST), incubated overnight at 4°C in biotinylated secondary antisera, washed twice in PBS and then incubated for 1 h in avidin-biotin-peroxydase complex (Vectastain Elite, Vector Laboratories). Following a final wash in PBS, the sections were developed in 1-naphtol ammonium carbonate solution [26]. Sections were then mounted on gelatin-coated slides and air dried. After a basic dye enhancement step in 0.025% Crystal violet (Aldrich, 42555) solution in phosphate-buffer (PB) for 3 min and two short PB rinses to take off the excess of stain, sections were differentiated in
Fig. 1. Photographs illustrating ubiquitin (upper row) and hsp 70 (lower row) immunoreactivityin alternate sections (40 pm thick) at lumbar level (Ls)of spinal cord of rats 14 days after loose ligationsof their common sciatic nerve (left side). Right side is used as a control sideto demonstratecharacteristicsof constitutiveimmunoreactivityfor each protein. Arrows point to dorsal group of cells; arrow heads to ventral motoneurones.
70% and 95% alcohol with the time differentiation to be evaluated under the microscope. Staining appeared as an intense blue-violet colour. Sections were finally air dried and coverslipped. All positive staining was abolished when omitting one of the immunoreagents in the staining sequence. Motoneurone staining for hsp 70 and ubiquitin are illustrated in Fig. 1. The lesioned side is shown on left, while the right side which corresponds to the intact nerve serves as control. As seen on the control side (right) both ubiquitin (upper row) and hsp 70 (lower row) are constitutively expressed in motoneurones. Staining for ubiquitin is normally moderate while that for hsp 70 is intense. Both intensities of staining are clearly modified in motor cells subject to axon damage (left side). The effects are maximal for cells located dorsally (arrows). Ventral cells (arrow heads), in contrast, show staining intensities similar to those seen on the control side. These differential effects depending on the group of motoneurones under study may result from differential compression of nerve axons in relationship with their locations within the nerve trunk. Staining was differentially altered for each protein. Hsp 70 labeling which is initially high in intact cells was clearly reduced; ubiquitin labeling which is normally moderate was greatly increased after lesion. Both of these observations suggest significant metabolic changes in stressed cells. In accordance with reports [9, 14, 18, 20, 23, 32] regarding other models of axon damage (cut, crush) lesion of axons additionally resulted in
51
increased stainings for c-JUN and rNGF. However and in accordance with information from other models of nerve lesioning [1, 12, 28, 29, 31] no differences could be detected for CGRP labeling in motoneurones on either the lesioned or non-lesioned side at those long survival period times. The present observations demonstrate that studying hsp 70 and ubiquitin contents can be an easy way of demonstrating metabolic stress in motoneurones undergoing axon damage and suggests that the abnormal motor behaviours that accompany neuropathy in response to loose ligation of the common sciatic nerve could be partly explained by motoneurone disturbance. Such motoneurone impairment could also be the origin of the muscle atrophy previously reported by Bennett and Xie [4]. The decrease in motoneurone hsp 70 content under these chronic injury conditions fits well with observations consecutive to axotomy [27] showing that following a few hours of transient increase, hsp 70 contents rapidly decline to levels below controls. Such hsp 70 depletion at the cell body level could be due to either a negative regulation of its gene transcription or to a local decrease due to axonal transport towards the lesion site for cytoskeleton stabilization [7]. In the present study alterations in hsp 70 contents accompany those of ubiquitin. Ubiquitin has been considered as a marker for neurodegenerative diseases [10] related to the accumulation of abnormal proteins rather to neuronal death [15, 19]. An interaction between ubiquitin and the heat shock transcription factor has been postulated [5] suggesting that hsp transcription is dependent on the cellular levels of ubiquitin. Regulation of hsp 70 transcription could also be under control of c-JUN protein as hsp gene promoter contains an activating transcription factor (ATF)binding site [13], and as ATF and c-JUN may dimerize to form active transcriptional factors [22]. The overexpression we have noted for c-JUN consequently could be in relationship to the changes in hsp 70 contents. On the other hand, it is highly probable that all the variations we have noted for hsp 70, ubiquitin and c-JUN result from the liberation of specific factors or damaged proteins at the site of the lesion. One possible factor could be the nerve growth factor (NGF) that is released by non-neuronal cells surrounding damaged axons [16]. The appearance of immunoreactivity for the receptor to N G F in parallel with changes in motoneurone contents of hsp 70, ubiquitin and c-JUN as we observed would strongly suggest a role for this trophic factor in triggering metabolic reactions. The authors are indebted to R Sanderson for her kindness in preparing the English text. They are grateful to A. Mendtrey and J. Corvalan for preparing the illustrations.
This study was supported by Institut National de la Sant6 et de la Recherche M6dicale (INSERM) funds. D.M. is supported by Centre de la Recherche Scientifique (CNRS), J.D. by a fellowship from Fonds National Suisse pour la Recherche Scientifique (FNSRS) and M.L.-M. by a fellowship from INSERM. 1 Arvidsson, U., Johnson, H., Piehl, F., Cullheim, S., H6kfelt, T., Risling, M., Terenius, L. and Ulfhake, B., Peripheral nerve section induces increased levels of calcitonin gene-related peptide (CGRP)like immunoreactivity in axotomized motoneurons, Exp. Brain Res., 79 (1990) 212-216. 2 Attal, N., Jazat, F., Kayser, V. and Guilbaud, G., Further evidence for 'pain-related' behaviours in a model of unilateral peripheral mononeuropathy, Pain, 41 (1990) 235-251. 3 Basbaum, A.I., Gautron, M., Jazat, F., Mayes, M. and Guilbaud, G., The spectrum of fiber loss in a model of neuropathic pain in the rat: an electron microscopic study, Pain, 47 (1991) 359-367. 4 Bennett, G.J. and Xie, Y.-K., A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man, Pain, 33 (1988) 87-107. 5 Burdon, R.H., Heat shock and the heat shock proteins, Biochem. J., 240 (1986) 313-324. 6 Chandler, C.E., Parsons, L.M., Hosang, M. and Shooter, E.M., A monoclonal antibody modulates the interaction of nerve growth factor with PC12 cells, J. Biol. Chem., 259 (1984) 6882-6889. 7 Clark, B.D. and Brown, I.R., Axonal transport of a heat shock protein in the rabbit visual system, Proc. Natl. Acad. Sci. USA, 82 (1985) 1281-1285. 8 Committee for Research and Ethical Issues of the IASE Ethical standards for investigations of experimental pain in animals, Pain, 16(1983) 109 110. 9 Ernfors, P., Henschen, A., Olson, L. and Persson, H., Expression of nerve growth factor receptor mRNA is developmentally regulated and increased after axotomy in rat spinal cord motoneurons, Neuron, 2 (1989) 1605-1613. 10 Gallo, J.M. and Anderton, B.H., Ubiquitous variations in nerves, Nature, 337 (1989) 687-688. 11 Gautron, M., Jazat, F., Ratinahirana, H., Hauw, J.J. and Guilbaud, G., Alterations in myelinated fibres in the sciatic nerve of rats after constriction: possible relationships between the presence of abnormal small myelinated fibres and pain-related behaviour, Neurosci. Lett., 111 (1990) 28-33. 12 Haas, C.A., Streit, W.J. and Kreutzberg, G.W., Rat facial motoneurons express increased levels ofcalcitonin gene-related peptide mRNA in response to axotomy, J. Neurosci. Res., 27 (1990) 270275. 13 Hal, T., Liu, F., Coukos, W.J. and Green, M.R., Transcription factor ATF cDNA clones: an extensive family of leucine zipper proteins able to selectively form DNA-binding heterodimers, Genes Dev., 3 (1989) 2083-2090. 14 Herdegen, T., Kummer, W., Fiallos, C.E., Leah, J. and Bravo, R., Expression of c-JUN, JUN B and JUN D proteins in rat nervous system following transection of vagus nerve and cervical sympathetic trunk, Neuroscience, 45 (1991) 413422. 15 Hershko, A., The ubiquitin pathway for protein degradation, Trends Biochem. Sci., 16 (1991) 265-268. 16 Heumann, R., Korsching, S., Bandtlow, C. and Thoenen, H., Changes of nerve growth factor synthesis in non-neuronal cells in response to sciatic nerve transection, J. Cell Biol., 104 (1987) 16231631.
52 17 Hightower, L.E., Heat shock, stress proteins, chaperones, and proteotoxicity, Cell, 66 (1991) 191-197. 18 Jenkins, R. and Hunt, S.P., Long-term increase in the levels of c-jun mRNA and Jun protein-like immunoreactivity in motor and sensory neurons following axon damage, Neurosci. Lett., 129 (1991) 107-110. 19 Johnson, E.S., Bartel, B., Seufert, W. and Varshavsky, A., Ubiquitin as a degradation signal, EMBO J., 11 (1992) 497-505. 20 Koliatsos, V.E., Crawford, T.O. and Price, D.L., Axotomy induces nerve growth factor receptor immunoreactivity in spinal motor neurons, Brain Res., 549 (1991) 297-304. 21 Kupers, R.C., Nuytten, D., De Castro-Costa, M. and Gybels, J.M., A time course analysis of the changes in spontaneous and evoked behaviour in a rat model of neuropathic pain, Pain, 50 (1992) 101111. 22 Lamb, P. and MacKnight, S.L., Diversity and specificity in transcriptional regulation: the benefits of heterotopic dimerization, Trends Biochem. Sci., 16 (1991) 417-422. 23 Leah, J.D., Herdegen, T. and Bravo, R., Selective expression of JUN proteins following axotomy and axonal transport block in peripheral nerves in the rat: evidence for a role in the regeneration process, Brain Res., 566 (1991) 198-207. 24 Manzerra, P. and Brown, I.R., Expression of heat shock genes (hsp 70) in the rabbit spinal cord: localization of constitutive and hyperthermia-inducible mRNA species, J. Neurosci. Res., 31 (1992) 606615. 25 Mazurkiewicz, J.E., Ubiquitin deposits are present in spinal motor neurons in all stages of the disease in the motor neuron degeneration (Mnd) mutant of the mouse, Neurosci. Lett., 128 (1991) 182186. 26 Menrtrey, D., de Pommery, J., Baimbridge, K.G. and Thomasset, M., Calbindin-D28K (CaBP28k)-like immunoreactivity in ascend-
27
28
29
30 31
32
33
34
ing projections. I. Trigeminal nucleus caudalis and dorsal vagal complex projections, Eur. J. Neurosci., 4 (1992) 61-69. New, G.A., Hendrickson, B.R. and Jones, K.J., Induction of heat shock protein 70 mRNA in adult hamster facial nuclear groups following axotomy of the facial nerve, Methods Brain Disease, 4 (1989) 273-279. Noguchi, K., Senba, E., Morita, Y., Sato, M. and Tohyarna, M., ct-CGRP and fl-CGRP mRNAs are differentially regulated in the rat spinal cord and dorsal root ganglion, Mol. Brain Res., 7 (1990) 299-304. Piehl, F., Arvidsson, U., Johnson, H., Cullheim, S., Villar, M., Dagerlind, A., Terenius, L., H6kfelt, T. and Ulfhake, B., Calcitonin gene-related peptide (CGRP)-like immunoreactivity and CGRP mRNA in rat spinal cord motoneurons after different types of lesions, Eur. J. Neurosci., 3 (1991) 737-757. Rechsteiner, M., Natural substrates of the ubiquitin proteolytic pathway, Cell, 66 (1991) 615-618. Saika, T., Senba, E., Noguchi, K., Sato, M., Kubo, T., Matsunaga, T. and Tohyama, M., Changes in expression of peptides in rat facial motoneurons after facial nerve crushing and resection, Mol. Brain Res., 11 (1991) 187-196. Saika, T., Senba, E., Noguchi, K., Sato, M., Yoshida, S., Kubo, T., Matsunaga, T. and Tohyama, M., Effects of nerve crush and transection on mRNA levels for nerve growth factor receptor in the rat facial motoneurons, Mol. Brain Res., 9 (1991) 157-160. Schiffer, D., Autilio-Gambetti, L., Chi6, A., Gambetti, P., Giordana, M.T., Gullotta, F., Migheli, A. and Vigliani, M.C., Ubiquitin in motor neuron disease: study at the light and electron microscope, J. Neuropathol. Exp. Neurol.0 50 (1991) 463-473. Wayne, D.B. and Heaton, M.B., The ontogeny of specific retrograde transport of nerve growth factor by motoneurons of the brainstem and spinal cord, Dev. Biol., 138 (1990) 484498.
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NSL 09422
Opposite effects of axon damage on heat shock proteins (hsp 70) and ubiquitin contents in motor neurons of neuropathic rats M. Lant6ri-Minet b, J.A. Desmeules c and D. Men6trey a aInstitut National de la Santd et de la Recherche Mddicale Unitd 161, Paris (France), bLaboratoire de Neuro-oncologie et Neurosciences Cliniques, Service de Neurologie du Centre Hospitalo-Universitaire de Nice, Nice (France) and CDivision de Pharmacologie Clinique, Hopital Cantonal Universitaire de Genkve,. GenOve (Switzerland) (Received 29 September 1992; Revised version received 13 December 1992; Accepted 23 December 1992 Key words: Nerve ligature; Chaperon; Proteolytic factor; c-JUN; Nerve growth factor receptor; Calcitonin gene-related peptide; Motoneuron; Immunocytochemistry Alteration in the motoneurone contents of heat shock protein (hsp 70) and ubiquitin were studied in rats which had been subject to loose ligation of one common sciatic nerve. This results in a unilateral peripheral neuropathy which peaks at 14 days following ligation and is characterized by transient degeneration of both myelinated and unmyelinated nerve fibres, abnormal motor behaviours (posture of the hind limb, walking patterns) and thermal and mechanical allodynia of the hind paw. Hsp 70 and ubiquitin are proteins involved in protein metabolism and their expression is regulated during cellular stress. The contralateral unlesioned side was used as control. Motoneurone staining for hsp 70 and ubiquitin were differentially altered at the peak of the neuropathy. Axon damage resulted in a decrease in hsp 70 labeling while ubiquitin staining increased. At the same time motoneurones undergoing axon damage overstained for the immediate early gene encoded protein c-JUN and for nerve growth factor receptor (rNGF). In contrast, no clear alteration was seen, at that time, in the intensity of labeling for calcitonin gene-related peptide (CGRP). This study demonstrates that peripheral neuropathy resulting from loose ligation of the common sciatic nerve not only produces sensory alterations as previously reported but also leads to pronounced alterations in motoneurone functioning that could partly explain the observed abnormal motor behaviours. Results are discussed in accordance with presumed roles for hsp 70 and ubiquitin in protein metabolism and in relationship with possible interaction with c-JUN and rNGF expressions.
Protein metabolism in vivo depends on intracellular proteins that are present either constitutively or expressed during physiological stress (see refs. in 5, 17 and 30). Among these proteins are the 70-kDa heat shock proteins (hsp 70) and ubiquitin which act as molecular chaperones or proteolytic factors, respectively. Expression of both constitutive and inducible hsp 70 proteins have been recently reported in the spinal cord [24]; constitutive proteins exist in grey matter areas including motor neurones while hyperthermia-inducible proteins primarily exist in white matter. Ubiquitin has been reported in motoneurones in cases of degenerative diseases [25, 33]. These observations suggest that both hsp 70 and ubiquitin are involved in the turn-over of proteins in motor cells, and that they could be useful markers in demonstrating functional alterations in motoneurone metabolism. Immunoreactivity for hsp 70 and ubiquitin
Correspondence: D. Men6trey, INSERM U 161, 2 rue d'Al6sia, 75014 Paris, France.
was consequently studied in order to evaluate possible motoneuronal alterations consecutive to unilateral peripheral neuropathy resulting from loose ligation of the common sciatic nerve [4]. This disorder which is characterized by transient degeneration of both myelinated and unmyelinated nerve fibers [3, 4, 11], abnormal motor behaviours (posture of the hind limb, walking patterns) and thermal and mechanical allodynia of the hind paw [2, 21] has become a very popular model for chronic pain studies of deafferentation. However, it has never been fully understood if the guarding positions and/or abnormal gait this disorder induces only reflect painful sensations, or are directly related to paresia as a result of the motor deficit. The immunohistochemical study of hsp 70 and ubiquitin contents of motoneurones may provide an easy way to evaluate the degree of the motoneurone impairment. Since motoneurone overexpression of the immediate early gene c-JUN [14, 18, 23], of calcitonin gene-related peptide (CGRP) [1, 12, 28, 29, 31], and of nerve growth factor receptor (rNGF) [9, 20, 32] has been previously noted following axon damage, possible alterations
50 in staining for all of these proteins were also studied in parallel. Ten adult male albino rats of the Sprague-Dawley strain were used. Unilateral ligations of the common sciatic nerve were performed under sodium pentobarbital anaesthesia (50 mg/kg i.p.) by four loose ligatures (5.0 chromic gut) spaced at 1-2 mm intervals. Ligatures are tied loosely to reduce so as not to interrupt blood circulation through the epineural vasculature. Sham procedures which consisted of similar surgery except for ligations were performed on the opposite side. Animals were kept unanaesthetized for the following survival period and managed in accordance with the guidelines of the ethical standards of the IASP committee [8]. Immunohistochemical studies were performed at the peak of the neuropathy (2 weeks). Animals were anaesthetized with sodium pentobarbital and perfused intracardially with 200 ml of 0.1 M phosphate-buffered saline (PBS), pH 7.4, followed by 400 ml of 1% paraformaldehyde in PBS. Tissue was then removed, immersed in 4% fixative solution for 3 h and cryoprotected overnight in phosphate-buffered 30% sucrose solution at 4°C. Frozen serial coronal sections (40 pm) were cut and collected in PBS to be processed as free-floating sections. Sections were incubated in normal serum (10% in phosphate-buffered saline and 0.3% Triton X-100 (PBST)) for 30 min followed by the primary antisera at 4°C overnight. The antisera to hsp 70 and r N G F were monoclonal; hsp 70 antiserum was purchased from Amersham (RPN 1197; Amersham, UK) and run at 1/10,000; r N G F antiserum (Igg 192) was a kind gift of Dr. E.M. Johnson (Washington University, St. Louis, USA) and run at 4/lg/ml. The r N G F antiserum is directed against the low-affinity form of the receptor [6]. The antisera to c-JUN, ubiquitin and C G R P were polyclonal; ubiquitin antiserum was purchased from Chemicon (AB 1690; Temecula, CA, USA) and run at 1/2000; C G R P antiserum was purchased from Cambridge Research Biochemicals (CA 08 220; Northwich, UK) and run at 1/10,000; c-JUN antiserum was a gift of Dr. R. Bravo (Bristol-Meyers Squidd, Princeton, USA) and run at 1/20,000. After incubation in primary antisera, sections were washed in three changes of normal goat serum (1% in PBST), incubated overnight at 4°C in biotinylated secondary antisera, washed twice in PBS and then incubated for 1 h in avidin-biotin-peroxydase complex (Vectastain Elite, Vector Laboratories). Following a final wash in PBS, the sections were developed in 1-naphtol ammonium carbonate solution [26]. Sections were then mounted on gelatin-coated slides and air dried. After a basic dye enhancement step in 0.025% Crystal violet (Aldrich, 42555) solution in phosphate-buffer (PB) for 3 min and two short PB rinses to take off the excess of stain, sections were differentiated in
Fig. 1. Photographs illustrating ubiquitin (upper row) and hsp 70 (lower row) immunoreactivityin alternate sections (40 pm thick) at lumbar level (Ls)of spinal cord of rats 14 days after loose ligationsof their common sciatic nerve (left side). Right side is used as a control sideto demonstratecharacteristicsof constitutiveimmunoreactivityfor each protein. Arrows point to dorsal group of cells; arrow heads to ventral motoneurones.
70% and 95% alcohol with the time differentiation to be evaluated under the microscope. Staining appeared as an intense blue-violet colour. Sections were finally air dried and coverslipped. All positive staining was abolished when omitting one of the immunoreagents in the staining sequence. Motoneurone staining for hsp 70 and ubiquitin are illustrated in Fig. 1. The lesioned side is shown on left, while the right side which corresponds to the intact nerve serves as control. As seen on the control side (right) both ubiquitin (upper row) and hsp 70 (lower row) are constitutively expressed in motoneurones. Staining for ubiquitin is normally moderate while that for hsp 70 is intense. Both intensities of staining are clearly modified in motor cells subject to axon damage (left side). The effects are maximal for cells located dorsally (arrows). Ventral cells (arrow heads), in contrast, show staining intensities similar to those seen on the control side. These differential effects depending on the group of motoneurones under study may result from differential compression of nerve axons in relationship with their locations within the nerve trunk. Staining was differentially altered for each protein. Hsp 70 labeling which is initially high in intact cells was clearly reduced; ubiquitin labeling which is normally moderate was greatly increased after lesion. Both of these observations suggest significant metabolic changes in stressed cells. In accordance with reports [9, 14, 18, 20, 23, 32] regarding other models of axon damage (cut, crush) lesion of axons additionally resulted in
51
increased stainings for c-JUN and rNGF. However and in accordance with information from other models of nerve lesioning [1, 12, 28, 29, 31] no differences could be detected for CGRP labeling in motoneurones on either the lesioned or non-lesioned side at those long survival period times. The present observations demonstrate that studying hsp 70 and ubiquitin contents can be an easy way of demonstrating metabolic stress in motoneurones undergoing axon damage and suggests that the abnormal motor behaviours that accompany neuropathy in response to loose ligation of the common sciatic nerve could be partly explained by motoneurone disturbance. Such motoneurone impairment could also be the origin of the muscle atrophy previously reported by Bennett and Xie [4]. The decrease in motoneurone hsp 70 content under these chronic injury conditions fits well with observations consecutive to axotomy [27] showing that following a few hours of transient increase, hsp 70 contents rapidly decline to levels below controls. Such hsp 70 depletion at the cell body level could be due to either a negative regulation of its gene transcription or to a local decrease due to axonal transport towards the lesion site for cytoskeleton stabilization [7]. In the present study alterations in hsp 70 contents accompany those of ubiquitin. Ubiquitin has been considered as a marker for neurodegenerative diseases [10] related to the accumulation of abnormal proteins rather to neuronal death [15, 19]. An interaction between ubiquitin and the heat shock transcription factor has been postulated [5] suggesting that hsp transcription is dependent on the cellular levels of ubiquitin. Regulation of hsp 70 transcription could also be under control of c-JUN protein as hsp gene promoter contains an activating transcription factor (ATF)binding site [13], and as ATF and c-JUN may dimerize to form active transcriptional factors [22]. The overexpression we have noted for c-JUN consequently could be in relationship to the changes in hsp 70 contents. On the other hand, it is highly probable that all the variations we have noted for hsp 70, ubiquitin and c-JUN result from the liberation of specific factors or damaged proteins at the site of the lesion. One possible factor could be the nerve growth factor (NGF) that is released by non-neuronal cells surrounding damaged axons [16]. The appearance of immunoreactivity for the receptor to N G F in parallel with changes in motoneurone contents of hsp 70, ubiquitin and c-JUN as we observed would strongly suggest a role for this trophic factor in triggering metabolic reactions. The authors are indebted to R Sanderson for her kindness in preparing the English text. They are grateful to A. Mendtrey and J. Corvalan for preparing the illustrations.
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