Response of facial and rubrospinal neurons to axotomy: changes in mRNA expression for cytoskeletal proteins and GAP-43.

Neurons confined within the mammalian CNS usually do not regenerate after axonal injury, while axonal regeneration is the rule in the PNS. It has been hypothesized that this may be related to differences in the microenvironment of the PNS versus CNS and to differences in the neuronal response to injury. In order to test the latter hypothesis, we compared changes in gene expression after axotomy in two populations of neurons: rat facial motoneurons and rat rubrospinal neurons. In situ hybridization with cDNA probes for the medium and light neurofilament protein revealed a reduced mRNA content in both facial and rubrospinal neurons at all times investigated (i.e., 1, 2, and 3 weeks after axotomy). On the other hand, mRNAs for actin and tubulin were increased in both neuronal populations during the first week after axotomy. While this increase was sustained in facial motoneurons for several weeks, total tubulin mRNA and actin mRNA were decreased in rubrospinal neurons at 2 and 3 weeks after axotomy, coincident with their atrophy. The developmentally regulated T alpha 1 tubulin mRNA, which was previously shown to be reexpressed in facial motoneurons after axotomy, was elevated severalfold in axotomized rubrospinal neurons, and increased levels persisted in some rubrospinal neurons as late as 7 weeks after axotomy. Similarly, the developmentally regulated GAP-43 mRNA increased in both axotomized facial and rubrospinal neurons, and increased levels were sustained in some axotomized rubrospinal neurons for at least 7 weeks. The response of rubrospinal neurons to axotomy in the cervical spinal cord is, in the first week, qualitatively similar to the response of facial motoneurons. However, by 2 weeks after axotomy there is a generalized reduction in mRNA levels for all three cytoskeletal proteins that is associated with neuronal atrophy. During this period, mRNA levels for the two specific markers of the growth state, T alpha 1 tubulin and GAP-43, remain elevated. Thus, axotomy of rubrospinal neurons appears to set in motion two independent events. First, an axotomy signal initiates a cell-body reaction similar to that of PNS neurons, including increased mRNA levels for T alpha 1 tubulin and GAP-43. Later, a generalized cellular atrophy and decrease in mRNA levels occur without reversing the specific responses of T alpha 1 and GAP-43 to axotomy. We conclude that the failure of rubrospinal neurons to regenerate is not due to a failure to initiate gene-expression changes characteristic of regenerating peripheral neurons.(ABSTRACT TRUNCATED AT 400 WORDS)     

Developmental regulation of GAP-43, glutamine synthetase and beta-actin mRNA in rat cortical astrocytes.

Steady-state levels of mRNA encoding growth-associated protein 43 (GAP-43), glutamine synthetase (GS) and beta-actin were measured during development of neonatal rat cortical astrocytes in primary culture. GAP-43 mRNA and protein decreased rapidly during the first 2 weeks and slowly thereafter. In contrast, GS mRNA increased approximately 3-fold during the first 2 weeks and reached maximum by day 15. Actin mRNA first increased up to 8 days and decreased thereafter reaching a constant amount of 15 days, similar to the initial low value. Thus, GAP-43, GS and beta-actin mRNA levels are differentially regulated during development of astrocytes in primary culture. Because the patterns of expression of astrocytic markers GS and GFAP (shown previously) in vitro and in vivo are similar to each other, primary cultures of astrocytes may be an excellent system for investigating mechanisms of developmental regulation of these genes.     

Role of GAP-43 in mediating the responsiveness of cerebellar and precerebellar neurons to axotomy

To determine whether the competence for axonal sprouting and/or regeneration in the cerebellar system correlates with GAP-43 expression, we have studied GAP-43 mRNA and protein expression in the postlesioned cerebellum and inferior olive. Purkinje cells transiently express GAP-43 during their developmental phase (from E15 to P5 in the rat) which consists of fast axonal growth and the formation of the corticonuclear projection. Adult Purkinje cells, which in control adult rats do not express GAP-43, are extremely resistant to the effects of axotomy but cannot regenerate axons. However, a late and protracted sprouting of axotomized Purkinje cells occurs spontaneously and correlates with a mild expression of GAP-43 mRNA. In contrast, inferior olivary neurons, despite their high constitutive expression of GAP-43, do not sprout but retract their axons and die after axotomy. Furthermore, mature Purkinje cells in cerebellar explants of transgenic mice that overexpress GAP-43 do not regenerate after axotomy, even in the presence of a permissive substrate (cerebellar embryonic tissue) and, contrary to the case in wild-type mice, they do not survive in the in vitro conditions and undergo massive cell death. These results show that the expression of GAP-43 is not only associated with axonal growth, but also with neuronal death.     

Treatment of chronically injured spinal cord with neurotrophic factors stimulates betaII-tubulin and GAP-43 expression in rubrospinal tract neurons

Exogenous neurotrophic factors provided at a spinal cord injury site promote regeneration of chronically injured rubrospinal tract (RST) neurons into a peripheral nerve graft. The present study tested whether the response to neurotrophins is associated with changes in the expression of two regeneration-associated genes, betaII-tubulin and growth-associated protein (GAP)-43. Adult female rats were subjected to a right full hemisection lesion via aspiration of the C3 spinal cord. A second aspiration lesion was made 4 weeks later and gel foam saturated in brain-derived neurotrophic factor (BDNF), glial cell-line derived neurotrophic factor (GDNF), or phosphate-buffered saline (PBS) was applied to the lesion site for 60 min. Using in situ hybridization, RST neurons were examined for changes in mRNA levels of betaII-tubulin and GAP-43 at 1, 3, and 7 days after treatment. Based on analysis of gene expression in single cells, there was no effect of BDNF treatment on either betaII-tubulin or GAP-43 mRNA expression at any time point. betaII-Tubulin mRNA levels were enhanced significantly at 1 and 3 days in animals treated with GDNF relative to levels in animals treated with PBS. Treatment with GDNF did not affect GAP-43 mRNA levels at 1 and 3 days, but at 7 days there was a significant increase in mRNA expression. Interestingly, 7 days after GDNF treatment, the mean cell size of chronically injured RST neurons was increased significantly. Although GDNF and BDNF both promote axonal regeneration by chronically injured neurons, only GDNF treatment is associated with upregulation of betaII-tubulin or GAP-43 mRNA. It is not clear from the present study how exogenous BDNF stimulates regrowth of injured axons.     

Naturally occurring cell death and differentiation of developing spinal motoneurons following axotomy

The purpose of this study was to examine the effects of axon transection on the development and differentiation of spinal motoneurons in the bullfrog (Rana catesbeiana) tadpole. The 3 ventral roots (VRs) that innervate the hindlimb were transected, and the animals were killed 6-7 weeks later (reinnervation took place within 3 weeks). At early stages of development, axotomy resulted in an increase in the number of spinal motoneurons on the operated side. By histological criteria, these motoneurons appeared more differentiated than those in normal tadpoles. Axotomy was effective in increasing motoneuron number only during the period of naturally occurring cell death. Similar effects were seen when the transected VRs were ligated to prevent regeneration. Hindlimb amputation without VR transection had no effect on motoneuron number or differentiation. Thus, target removal is neither a necessary nor a sufficient condition for hyperplasia of the lateral motor column. An extreme loss of spinal motoneurons was seen if the operated tadpole entered into metamorphic climax during the 6-7-week postoperative survival period. Motoneuron loss occurred although the injured motoneurons had reconnected to the hindlimb. In contrast, tadpoles allowed to survive up to 6 months showed no loss of motoneurons if they did not enter metamorphic climax. From these data, it appears axon transection in developing spinal motoneurons exerts its effects on motoneuron number and differentiation by altering the metabolic state of the motoneuron (axon reaction) rather than by depriving it of contact with its target.     

Estrogenic regulation and sex dimorphism of growth-associated protein 43 kDa (GAP-43) messenger RNA in the rat.

In addition to effects on brain protein synthesis, neurotransmitter release, and electrophysiology, estrogens alter neurite outgrowth and synaptogenesis. This study examined in the adult rat the effects of estrogen and sex on the expression of the GAP-43 gene; encoding a phosphoprotein structurally and physiologically linked to these two processes in the rat CNS. Ovariectomized (OVX) rats were injected with vehicle or estrogen, or male and female rats were either gonadectomized or left intact. Brains were dissected to obtain ventromedial hypothalamus (VMH), posterior hypothalamus (PH), or frontal cortex (CTX). Total RNA from these areas were extracted, and slot-blots of equal masses of total RNA were hybridized to 32P-labeled cDNAs for GAP-43 and beta-actin, and also to synthetic poly-dT. Resultant autoradiograms were scanned by laser densitometry, quantitated, and ratios of the gray scale generated by each probe were compared between experimental groups. GAP-43 mRNA expression, when compared to expression of either beta-actin mRNA or total poly(A)-containing RNA (poly(A) RNA), was higher in VMH and PH as compared to CTX. Estrogen treatment of OVX rats resulted in a 48-74% increase in GAP-43 mRNA levels in the VMH--in one experiment, this increase was noted after 2 h of estradiol treatment, and in another after 3 days of estradiol benzoate treatment; but PH and CTX were unaffected by either estrogen regimen. Conversely, ovariectomy of intact rats decreased GAP-43 mRNA expression by 45% in the VMH, but not in the CTX.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulatory effects of trophic factors on expression and distribution of CGRP and GAP-43 in rat motoneurons

Trophic factors play important roles for the regulation of several motoneuron characteristics. In this report, we have studied the effects of different trophic factors on the regulation of two substances that have been linked with axon sprouting and regeneration in rat spinal motoneurons, i.e., calcitonin gene-related peptide (CGRP) and growth-associated protein-43 (GAP-43). The expression of both GAP-43 and CGRP is regulated developmentally and after different lesions. In the adult animal a sciatic nerve transection normally leads to an increased expression of both alpha-CGRP and GAP-43. Local administration of insulin-like growth factor-1 (IGF-1), IGF-2, or ciliary neurotrophic factor (CNTF) did not significantly change this response, whereas basic fibroblast growth factor (bFGF) attenuated the lesion-induced upregulation of alpha-CGRP mRNA. None of the studied factors had any influence on the upregulation of GAP-43 after lesion. In vitro, GAP-43 and alpha-CGRP mRNAs could be detected in enriched motoneuron cultures prepared from E14 rat embryos. The GAP-43 expression was upregulated by bFGF, IGF-1, IGF-2, and CNTF. CNTF also upregulated alpha-CGRP mRNA in cultured motoneurons, whereas bFGF had the opposite effect. IGF-1 and -2 did not significantly affect the alpha-CGRP mRNA levels. The decrease in GAP-43-immunopositive neuromuscular junctions (NMJs), seen during normal postnatal development, was attenuated after intramuscular injections of bFGF, CNTF, IGF-2, or CGRP 8–37, a CGRP antagonist. At the same time bFGF decreased and CNTF increased the number of CGRP-immunoreactive NMJs, whereas no difference from control was seen in IGF-2-treated muscles. These results show important regulatory effects of trophic factors on the expression of both GAP-43 and alpha-CGRP in motoneurons. However, it also is shown that the trophic factors used here influence the expression of these two substances in distinctively different patterns. This may have important implications for the understanding of trophic interactions in the spinal motor system and also should be considered in the evaluation of possible effects of CGRP and GAP-43 in motoneurons. The effect of a CGRP antagonist on GAP-43 in muscle indicates a role for CGRP in sprouting-related processes.     

Evidence for down-regulation of GAP-43 mRNA in Wobbler mouse spinal motoneurons by corticosterone and a 21-aminosteroid

Expression of the growth-associated protein GAP-43 is increased in the spinal cord of ALS patients and Wobbler (wr) mice, murine models of the disease. In this work we examined if expression of GAP-43 mRNA in control and wr mice was sensitive to steroid treatment. A group of control and wr mice received s.c. a 50 mg pellet of the natural hormone corticosterone (CORT) or the antioxidant 21-aminosteroid U-74389F during 4 days. Basal levels of GAP-43 mRNA were 10-fold elevated in ventral horn motoneurons of untreated wr mice, compared to the low levels in controls. The high expression of GAP-43 mRNA in wr was attenuated by treatment with CORT (41%, p<0.001) and U-74389F (36%, p<0.001). Although specific GAP-43 mRNA labelling was present in some neurons around the central canal, its cellular expression was similar in controls and wr. Also, steroid treatment was ineffective in neurons around the central canal. Other regions of the spinal cord (i.e., dorsal horn neurons) expressed GAP-43 mRNA slightly above background levels. It is possible that attenuation of GAP-43 expression due to the natural hormone and the antioxidant steroid resulted from reversal of motoneuron degeneration or aberrant sprouting. Therefore, steroid therapy may be of value to prevent denervation and/or muscular atrophy in this animal model.     

Alterations to neuronal polarity following permanent axotomy: a quantitative analysis of changes to MAP2a/b and GAP-43 distributions in axotomized motoneurons in the adult cat

Following axotomy, morphologically unusual, distal processes (UDPs) emerge from motoneuron dendrites. These processes contain an axonal protein, growth‐associated protein 43 (GAP‐43) but lack immunostaining for the dendritic protein microtubule‐associated protein 2a/b (MAP2a/b). Thus, it appears that neuronal polarity alters following axotomy. Our goal was to describe this change in neuronal polarity on a more detailed and quantitative level. We asked two questions: Following axotomy, where in the entire neuron does the immunoreactivity for MAP2a/b and GAP‐43 change and do these changes reflect a transformation of dendrite to axon or growth from terminal dendrites? Using intracellular labeling and immunocytochemistry, changes in MAP2a/b and GAP‐43 immunoreactivity were also found in processes with a morphology typical of terminal branches of intact motoneurons (called simple distal processes [SDPs]), as well as UDPs. Trajectories (the path from the soma to a single terminus) with UDPs and SDPs were longer than trajectories without these processes, and trajectories with UDPs were the longest. Trajectories without UDPs or SDPs were similar in length to trajectories from intact motoneurons. The distance from the soma to the point where MAP2a/b immunoreactivity became absent in trajectories with UDPs or SDPs was similar to the length of trajectories from intact motoneurons. Thus, following axotomy, two morphologically distinct types of axon‐like processes emerge from dendrites. The formation of these processes does not involve a transformation of the original dendrite, but rather growth at the ends of dendrites. J. Comp. Neurol. 450:318–333, 2002. © 2002 Wiley‐Liss, Inc.     

Effect of proximal axotomy on GAP-43 expression in cortical neurons in the mouse

As an approach to understanding why central neurons fail to regenerate, we have studied the response to proximal axotomy of transcallosal neurons of the cerebral cortex of the mouse. Anatomical studies have indicated only very slight regenerative responses by this population of cortical neurons. To further examine the regenerative response of these cells, we have looked by in situ hybridization at the expression of GAP-43 mRNA following axotomy caused by a stab wound delivered within about 200 μm to 1.25 mm of the cell body. Axotomized transcallosal neurons were compared with near-by unaxotomized transcallosal neurons, as well as with distant unaxotomized cortical neurons in the contralateral hemisphere. All three populations of neurons had been pre-labeled with Fluoro-Gold to allow identification. No up-regulation of GAP-43 mRNA above background levels was detected for axotomized cortical neurons at 1, 3 or 7 days after injury. In contrast, increases in mean silver grain density of up to 8-fold were measured in axotomized spinal cord motor neurons used as positive controls. Thus, as a population, the transcallosal cortical pyramidal neurons did not show a significant regenerative response, as monitored by GAP-43 upregulation, even with very close axotomy. These results identify this population of neurons as among the least regenerative studied, and suggest that, on a molecular level, inherent neuronal properties play a role in the limited regenerative response to brain injury.     

Overexpression of GAP-43 induces prolonged sprouting and causes death of adult motoneurons.

In neurodegenerative diseases, neurons undergo prolonged periods of sprouting. Whether this sprouting compromises these neurons is unknown. Here, we examined the effect of axotomy on adult motoneurons undergoing prolonged sprouting in transgenic mice that overexpress GAP-43 (growth-associated protein). Sciatic nerve injury in these adult mice results in motoneuron death, but has no effect in non-transgenic mice. Thus, continued growth of motor axons renders adult motoneurons susceptible to nerve injury and compromises their long-term survival. The progressive nature of neurodegenerative diseases may therefore be caused by prolonged sprouting.     

The response of rubrospinal neurons to axotomy in the adult opossum, Didelphis virginiana

To provide endpoints for developmental studies of rubrospinal plasticity in the North American opossum, we have attempted to determine the degree to which rubrospinal neurons survive axotomy in the adult animal. Bilateral or unilateral injections of the long-lasting fluorescent marker fast blue were made into the T-10 or the T-11 segment of the spinal cord to label rubrospinal neurons, and 7 days later, the rubrospinal tract was cut unilaterally four segments rostral to the injection(s). In cases with unilateral injections, the lesion was made ipsilateral to the injection. The animals were allowed to survive for 30-60 days before being sacrificed and perfused so that sections through the red nuclei could be examined for labeled neurons. The results show that most axotomized neurons survived the lesion, suggesting that lesion-dependent cell death is not a major factor in the failure of the rubrospinal tract to regenerate in the adult animal.     

Androgenic regulation of gap junctions between motoneurons in the rat spinal cord

Gap junctional plaques were found between androgen-sensitive motoneurons of the spinal nucleus of the bulbocavernosus (SNB), using thin-section and freeze-fracture techniques. The somata and proximal dendrites of SNB motoneurons were studied after retrograde labeling with cholera toxin conjugated to HRP. Of the gap junctions observed, 45% were somatodendritic, 35% were dendrodendritic, and 20% were somatosomatic. The removal of testosterone by castration dramatically reduced the number and diameter of the junctional plaques, and these changes were prevented by testosterone treatment. The results are the first demonstration of hormonal regulation of morphologically identified gap junctions in the CNS. The occurrence and hormonal dependence of gap junctional plaques between motoneurons of the lumbar spinal cord indicate that androgens regulate the degree of coupling between these cells, which may allow for the synchronization and amplification of the electrical activity in the nucleus.     

Astrocyte reactivity influences the number of presynaptic terminals apposed to spinal motoneurons after axotomy

Although synaptic plasticity is a widespread phenomenon, the underlying mechanisms leading to its occurrence are virtually unknown. In this sense, glial cells, especially astrocytes, may have a role in network changes of the nervous system, influencing the retraction of boutons as well as providing a proper perisynaptic environment, thereby affecting the replacement of inputs. Interestingly, the glial reaction does vary between strains of rats and mice. In this sense, we present evidence that C57BL/6J and A/J isogenic mice present different astrocyte reactivity after a peripheral lesion in vivo as well as in vitro, by analyzing primary cell cultures. Such a difference in the glial reaction has a direct influence on in vivo number of pre-synaptic terminals and on in vitro synaptogenesis.     

Growth-related changes in cell body size and succinate dehydrogenase activity of spinal motoneurons innervating the rat soleus muscle

Cell body sizes and oxidative enzyme (succinate dehydrogenase) activities of spinal motoneurons innervating the soleus muscle were determined in rats ranging in postnatal age from 3 to 13 weeks. The soleus motoneurons were labeled by a retrograde neuronal tracer, nuclear yellow. The mean cell body sizes of motoneurons increased from 3 to 7 weeks of age, while the mean succinate dehydrogenase activities of motoneurons decreased from 3 to 7 weeks of age. There were no changes in mean cell body size or mean succinate dehydrogenase activity of motoneurons from 7 to 13 weeks of age. An inverse relationship between cell body size and succinate dehydrogenase activity of motoneurons was observed, irrespective of age. These results indicate that motoneurons innervating the rat soleus muscle show the adult pattern of cell body size and succinate dehydrogenase activity at an earlier stage of postnatal growth, 7 weeks of age.     

Auditory brainstem: development and plasticity of GAP-43 mRNA expression in the rat.

Expression of the growth and plasticity associated protein GAP-43 is closely related to synaptogenesis and synaptic remodeling in the developing as well as in the mature nervous system. We have studied the postnatal development of GAP-43 mRNA expression in the auditory brainstem and determined the time course of its reexpression following deafening through cochlear ablation using a digoxigenin-coupled mRNA probe. By the first postnatal day, GAP-43 mRNA was expressed at high levels in all auditory brainstem nuclei. But whereas GAP-43 mRNA is almost entirely lost in most of these nuclei in the adult animal, significant levels of this molecule are retained in the inferior colliculus and, most notably, in the lateral and medial superior olivary nucleus. As a consequence of unilateral cochleotomy, GAP-43 mRNA rose dramatically in some neurons of the ipsilateral lateral superior olive, whereas the hybridization signal decreased in others. Using double staining protocols, we found that those olivary neurons that increase their level of GAP-43 mRNA appear to be identical with the cells developing strong GAP-43 immunoreactivity after cochleotomy. By combining axonal tracing with in situ hybridization, we proved that at least some of the cells with increased levels of GAP-43 mRNA and protein are the cells of origin of olivocochlear projections. A substantial decrease of the level of GAP-43 mRNA took place in the inferior colliculus contralateral to the lesioned cochlea. Our results led us to suggest that neurons in the superior olivary complex may play a crucial role in orchestrating auditory brainstem plasticity.     

Acute ethanol administration decreases GAP-43 and phosphorylated-GAP-43 in the rat hippocampus

Acute alcohol ingestion is well known to have deleterious effects on memory and also known to inhibit long-term potentiation, a putative cellular substrate of memory. In this study, we for the first time revealed that growth-associated protein 43 (GAP-43), which is well known as a presynaptic substrate of protein kinase C and one of the major synaptic plasticity-related genes, was down regulated by single ethanol administration (2.5 g/kg, 15% in saline, i.p.) in the rat hippocampus. Using real-time PCR, we confirmed that GAP-43 mRNA level is significantly decreased 2 h after ethanol administration. GAP-43 and p-GAP-43 (Ser41) immunoreactivities in the hippocampus were also reduced 4 h after ethanol administration. Immunohistochemical study showed that the reduction of GAP-43 and p-GAP-43 expression was associated with the perforant and mossy fibers pathways. These results suggest that the reduction of GAP-43 in the hippocampus might be, at least in part, a cause of memory impairment after acute ethanol ingestion.     

Neurite outgrowth and GAP-43 mRNA expression in cultured adult rat dorsal root ganglion neurons: Effects of NGF or prior peripheral axotomy

Adult dorsal root ganglion (DRG) cells are capable of neurite outgrowth in vivo and in vitro after axotomy. We have investigated, in cultured adult rat DRG cells, the relative influence of nerve growth factor (NGF) or a prior peripheral nerve lesion on the capacity of these neurons to produce neurites. Since there is evidence suggesting that the growth-associated protein GAP-43 may play a crucial role in axon elongation during development and regeneration, we have also compared the effect of these treatments on GAP-43 mRNA expression. NGF increased the early neurite outgrowth in a subpopulation of DRG cells. This effect was substantially less, however, than that resulting from preaxotomy, which initiated an early and profuse neurite outgrowth in almost all cells. No difference in the expression of GAP-43 mRNA was found between neurons grown in the presence or absence of NGF over 1 week of culture, in spite of the increased growth produced by NGF. In contrast, cultures of neurons that had been preaxotomized showed substantial increase in GAP-43 mRNA and NGF had, as expected, a significant effect on substance P mRNA levels. Two forms of growth may be present in adult DRG neurons: an NGF-independent, peripheral nerve injury-provoked growth associated with substantial GAP-43 upregulation, and an NGF-dependent growth that may underlie branching or sprouting of NGF-sensitive neurons, but which is not associated with increased levels of GAP-43 mRNA. © 1994 Wiley-Liss, Inc.     

GAP-43 mRNA localization in the rat hippocampus CA3 field.

Gene expression of the axonal growth-associated protein, GAP-43, has been studied in the adult rat brain by in situ hybridization histochemistry. This protein is synthesized at high levels in neuronal somata in immature and regenerating neurons, but after establishment of mature synaptic relations its synthesis generally declines sharply, thus providing a marker denoting propensity for exhibiting synaptic plasticity. Detailed examination of the distribution of mRNA for GAP-43 in rat hippocampus is selectively and robustly expressed in the pyramidal neurons of field CA3 and, to a lesser extent, the polymorph neurons of the hilus of the dentate gyrus. Additional hippocampal regions of moderate expression include the tenia tecta and the subicular and entorhinal fields, but CA1 and CA2 are strikingly lower in signal. The significance of this pattern of localization is considered in the context of the phosphorylation of GAP-43 and its role in influencing synaptic events underlying the establishment and maintenance of long-term potentiation and plasticity in the hippocampus.