Cell Body Response Research Articles

Chronic nerve compression injury induces a phenotypic switch of neurons within the dorsal root ganglia

Chronic nerve compression (CNC) injury initiates a series of pathological changes within the peripheral nerve at the site of injury. However, to date, little work has been performed to explore neuronal cell body responses to CNC injury. Here we show a preferential upregulation of growth-associated protein-43 (GAP-43) and enhanced Fluoro Ruby uptake by the small-diameter calcitonin gene-related protein (CGRP) and isolectin B4 (IB4)-positive neurons in the L4 and L5 ipsilateral dorsal root ganglion (DRG) 2 weeks and 1 month post injury. Furthermore, L4 and L5 DRGs ipsilateral to CNC injury also demonstrated a marked reduction in neurofilament 200 (NF-200) neurons and an increase in CGRP and IB4 neurons at early time points. All numbers normalized to values comparable to those of control when the DRG was evaluated 6 months post injury. Quantification of glial-derived neurotrophic factor (GDNF) protein revealed an upregulation in L4 and L5 DRG followed by a return to baseline values at later stages following injury. Upregulation of GDNF expression by Schwann cells was also readily apparent with both immunohistochemistry and Western blot analysis of 1 month compressed sciatic nerve specimens. Thus, CNC induces a phenotypic change in the DRG that appears to be temporally associated with increases in GDNF protein expression at and near the site of the compression injury in the nerve.

Activating transcription factor 3 induction in sympathetic neurons after axotomy: Response to decreased neurotrophin availability

Activating transcription factor 3 (ATF3) is induced in a high proportion of axotomized sensory and motor neurons after sciatic nerve transection. In the present study, we looked at the expression of this factor in the superior cervical ganglion (SCG) after axotomy and after other manipulations that induce certain aspects of the cell body response to axotomy. Sympathetic ganglia from intact rats and mice exhibit only a very occasional neuronal nucleus with activating transcription factor 3–like immunoreactivity (ATF3-IR); however, as early as 6 h and as late as 3 weeks postaxotomy, many of the neurons showed intense ATF3-IR. A second population of cells had smaller and generally less intensely stained nuclei, and at least some of these cells were satellite cells. Lesions distal to the SCG induced by administration of 6-hydroxydopamine or unilateral removal of the salivary glands produced increases in ATF3-IR similar to those seen after proximal axotomy, indicating that this response is not strictly dependent on the distance of the lesion from the cell body. Two proposed signals for triggering ATF3 expression were examined: reduction in nerve growth factor (NGF) availability and induction of the cytokine leukemia inhibitory factor (LIF). While administration of an antiserum raised against NGF to intact animals induced ATF3-IR, induction of ATF3-IR after axotomy was not reduced in LIF null mutant mice. Since axotomy, 6-hydroxydopamine, and sialectomy are known to decrease the concentration of NGF in the SCG, our data suggest that these decreases in NGF lead to increases in ATF3-IR. Furthermore, since the number of neurons in the SCG expressing ATF3-IR was greater after axotomy than after antiserum against NGF treatment, this raises the possibility that decreased NGF is not the only process regulating ATF3 expression after axotomy.

The strange case of Purkinje axon regeneration and plasticity

In the last few years Purkinje cells have become a most interesting model to investigate cellular/molecular mechanisms of axon regeneration and plasticity. Adult Purkinje cells are most peculiar for their weak cell body response to axotomy, which is accompanied by a strong resistance to injury and a virtually absolute inability to regenerate severed neurites, even in the presence of favourable environmental conditions. The same neurons show a vigorous intrinsic inclination toward axonal sprouting and structural plasticity, which can be elicited by removing extrinsic growth-inhibitory cues. These features gradually develop during early postnatal life, but the underlying mechanisms and biological significance remain unclear. This article reviews recent studies aimed at addressing these questions with respect to the general issue of brain repair. Indeed, understanding the reasons for the extremely poor regenerative capacity of Purkinje cells will be most important to elucidate basic biological mechanisms of axon regeneration and plasticity, and to promote circuit rewiring in the adult CNS.

Tumor necrosis factor‐α receptor 1 (p55) knockout only transiently decreases the activation of c‐Jun and does not affect the survival of axotomized dopaminergic nigral neurons

The activation of the c-Jun N-terminal kinases and their substrate transcription factor c-Jun is central to the death of dopaminergic neurons of the substantia nigra pars compacta (SNC) but the underlying signal cascades are poorly understood. We have studied the impact of the p55 tumor necrosis factor-alpha receptor (TNF-R) 1 on the N-terminal phosphorylation of c-Jun and the survival of the dopaminergic SNC neurons after transection of the medial forebrain bundle. The axotomy raised the immunoreactivities of tumor necrosis factor-alpha, p75 TNF-R2 and ED1 (ectodysplasin A) in the substantia nigra equally in wildtype and knockout (ko) mice and of TNF-R1 in wildtype mice. Importantly, TNF-R1 ko significantly reduced the early phosphorylation of c-Jun between 18 h and 3 d post-axotomy but the functional deficiency of TNF-R1 did not affect the survival of the dopaminergic neurons up to day 30. These findings demonstrate that: (i) TNF-R1 is involved in the early cell body response after axon transection; (ii) TNF-R1 operates upstream of c-Jun N-terminal kinase/c-Jun, the central signal system of nerve fiber injury, and (iii) the failure of persistent reduction of activated c-Jun is linked to the failure of protection of dopaminergic SNC neurons by TNF-R1 ko.

A conditioning lesion enhances sympathetic neurite outgrowth

Axonal regeneration can be influenced by a conditioning lesion (an axonal injury made prior to a second test lesion). Previously, sympathetic neurons in vivo were shown to respond to a conditioning lesion with decreased neurite outgrowth, in contrast to the enhanced outgrowth observed in all other peripheral neurons examined. The present experiments tested the effects of a conditioning lesion on neurite outgrowth in vitro from the superior cervical ganglion (SCG) and the impact of several factors on that response. Ganglia axotomized 1 week earlier and then explanted in Matrigel or collagen gel responded with a significant increase in neurite extension compared to sham-operated ganglia. A distal axotomy produced by unilateral removal of the salivary glands (sialectomy) caused an increase in neurite outgrowth similar to that of a proximal axotomy. These conditioning lesions induced both an increase in the rate of elongation, and, in the case of the proximally axotomized SCG, a shorter initial delay of outgrowth. The enhanced outgrowth following sialectomy was specific to the nerve containing the majority of axons projecting to the salivary glands, suggesting that the conditioning lesion effect is restricted to previously injured neurons. Deletion of the gene for leukemia inhibitory factor (LIF), a gene induced by axotomy, did not abolish the conditioning lesion effect in SCG explants or dissociated cell cultures. In conclusion, sympathetic neurons are capable of responding to a conditioning lesion with increased neurite outgrowth. The hypothesis that the neuronal cell body response to axotomy plays an important role in the conditioning lesion response is discussed.

Vimentin-Dependent Spatial Translocation of an Activated MAP Kinase in Injured Nerve

How are phosphorylated kinases transported over long intracellular distances, such as in the case of axon to cell body signaling after nerve injury? Here, we show that the MAP kinases Erk1 and Erk2 are phosphorylated in sciatic nerve axoplasm upon nerve injury, concomitantly with the production of soluble forms of the intermediate filament vimentin by local translation and calpain cleavage in axoplasm. Vimentin binds phosphorylated Erks (pErk), thus linking pErk to the dynein retrograde motor via direct binding of vimentin to importin beta. Injury-induced Elk1 activation and neuronal regeneration are inhibited or delayed in dorsal root ganglion neurons from vimentin null mice, and in rats treated with a MEK inhibitor or with a peptide that prevents pErk-vimentin binding. Thus, soluble vimentin enables spatial translocation of pErk by importins and dynein in lesioned nerve.

Axonal reinjury reveals the survival and re-expression of regeneration-associated genes in chronically axotomized adult mouse motoneurons

Recently, we reported that chronically axotomized rubrospinal neurons survive for up to 1 year in an atrophied state. This finding contrasted previous work suggesting the death of up to 50% of the neurons over time. In the adult mouse, the majority of facial motoneurons appear to be lost as a result of chronic nerve resection. Here, we sought to determine if chronically resected adult mouse facial motoneurons, like rubrospinal neurons, survive in an atrophied state. To test this hypothesis, we asked whether a second nerve injury, 10 weeks after an initial nerve resection, could stimulate a regenerative cell body response. After chronic resection (10 weeks), mouse facial motoneurons underwent atrophy resulting in a loss of countable neuronal cell bodies. In addition, the motoneurons failed to maintain their initial increase in expression of GAP-43 and α-tubulin mRNA. Reinjury of 10-week chronically resected facial motoneurons by the removal of the neuroma reversed the atrophy of the cell bodies and increased the percentage of identifiable cell bodies from 36% of contralateral to 79% in C57BL/6-C3H mice and from 28% of contralateral to 40% in Balb/c mice. Moreover, the reinjured motoneurons displayed an increase in GAP-43 and α-tubulin mRNA expression. The results of this study indicate that a second axon injury stimulates regenerative cell body responses in chronically resected mouse facial motoneurons and suggest previous studies using this model may have overestimated the number of dying motoneurons.

Axonal reinjury reveals the survival and re-expression of regeneration-associated genes in chronically axotomized adult mouse motoneurons

Recently, we reported that chronically axotomized rubrospinal neurons survive for up to 1 year in an atrophied state. This finding contrasted previous work suggesting the death of up to 50% of the neurons over time. In the adult mouse, the majority of facial motoneurons appear to be lost as a result of chronic nerve resection. Here, we sought to determine if chronically resected adult mouse facial motoneurons, like rubrospinal neurons, survive in an atrophied state. To test this hypothesis, we asked whether a second nerve injury, 10 weeks after an initial nerve resection, could stimulate a regenerative cell body response. After chronic resection (10 weeks), mouse facial motoneurons underwent atrophy resulting in a loss of countable neuronal cell bodies. In addition, the motoneurons failed to maintain their initial increase in expression of GAP-43 and α-tubulin mRNA. Reinjury of 10-week chronically resected facial motoneurons by the removal of the neuroma reversed the atrophy of the cell bodies and increased the percentage of identifiable cell bodies from 36% of contralateral to 79% in C57BL/6-C3H mice and from 28% of contralateral to 40% in Balb/c mice. Moreover, the reinjured motoneurons displayed an increase in GAP-43 and α-tubulin mRNA expression. The results of this study indicate that a second axon injury stimulates regenerative cell body responses in chronically resected mouse facial motoneurons and suggest previous studies using this model may have overestimated the number of dying motoneurons.

Activation of the c-Jun transcription factor following neurodegeneration in vivo

Neurodegenerative diseases often result in neuronal cell death, but the molecular mechanisms responsible are not fully understood. The expression and activation by phosphorylation of the c-Jun transcription factor plays an important role for the fate of affected neurons in response to injury. c-Jun is phosphorylated at serines 63 and 73 by the c-Jun N-terminal kinases and c-Jun N-terminal phosphorylation augments the transcriptional activity of c-Jun. Two approaches in neurodegeneration were investigated: The transection of the medial forebrain bundle to study neuronal cell body response in the derived neuronal populations of the substantia nigra pars compacta (SNC). This model of central axotomy leads as a long-term reaction to degeneration of the affected SNC neurons. A central component of the axotomy-induced alterations leading to neuronal degeneration is the rapid induction, lasting expression and activation of the c-Jun transcription factor. The focal cerebral ischemia, induced by occlusion of the arteria cerebri media and the subsequent reperfusion, serves as a suitable in vivo model for stroke. Also, ischemia leads to upregulation and activation of c-Jun and its target genes. Here the key role of c-Jun for the fate of neurons following degeneration is discussed with data received from experiments performed in Manfred Zimmermann's department investigating the effects of c-Jun on its target genes and on factors influencing c-Jun expression and activation.

Effects of fimbria-fornix transection on calpain and choline acetyl transferase activities in the septohippocampal pathway

The ability of fimbria-fornix bilateral axotomy to elicit calpain and caspase-3 activation in the rat septohippocampal pathway was determined using antibodies that selectively recognize either calpain- or caspase-cleaved products of the cytoskeletal protein αII-spectrin. Radioenzymatically determined choline acetyl transferase (ChAT) activity was elevated in the septum at day 5, but reduced in the dorsal hippocampus at days 3, 5 and 7, after axotomy. Prominent accumulation of calpain-, but not caspase-3-, cleaved spectrin proteolytic fragments was observed in both the septum and dorsal hippocampus 1–7 days after axotomy. ChAT-positive neuronal cell bodies in the septum also displayed calpain-cleaved spectrin indicating that calpain activation occurred in cholinergic septal neurons as a consequence of transection of the septohippocampal pathway. Calpain-cleaved αII-spectrin immunoreactivity was observed in cholinergic fibers coursing through the fimbria-fornix, but not in pyramidal neurons of the dorsal hippocampus, suggesting that degenerating cholinergic nerve terminals were the source of calpain activity in the dorsal hippocampus following axotomy. Accumulation of calpain-cleaved spectrin proteolytic fragments in the dorsal hippocampus and septum at day 5 after axotomy was reduced by i.c.v. administration of two calpain inhibitors. Calpain inhibition partially reduced the elevation of ChAT activity in the septum produced by transection but failed to decrease the loss of ChAT activity in the dorsal hippocampus following axotomy. These findings suggest that calpain activation contributes to the cholinergic cell body response and hippocampal axonal cytoskeletal degradation produced by transection of the septohippocampal pathway.

Changes in peptidyl-prolyl cis/trans isomerase activity and fk506 binding protein expression following neuroprotection by fk506 in the ischemic rat brain

FK506 is an immunosuppressant also showing neuroprotection following cerebral ischemia. FK506 binds to intracellular proteins (FKBP) which have a wide range of functions but have in common the peptidyl-prolyl cis/trans isomerase activity. Following transient focal ischemia, we have analyzed the expression of FKBP12, 52 and 65 and the total FKBP enzyme activity. Furthermore, we have investigated the effect of FK506 on signal transduction in neurons and perfusion changes in the infarct area.After 90 min of transient middle cerebral artery occlusion in male rats the expression of FKBP12, 52 and 65 was analyzed by Western blot in FK506-treated and control animals and the peptidyl-prolyl cis/trans isomerase activity was determined. Magnetic resonance imaging was used to measure tissue perfusion, development of vasogenic edema and infarct size. To investigate the neuronal stress signal cascade, activating transcription factor 2 (ATF-2), Fas-ligand (Fas-L) and c-Jun expression and phosphorylation were analyzed by immunohistochemistry.FK506 decreased the cerebral infarct volume by 53% and reduced the cytotoxic edema. The total FKBP enzymatic activity in the infarct area was increased and blocked dose dependently by FK506. FKBP expression was selectively up-regulated by cerebral ischemia. FK506 treatment does not influence the expression patterns. c-Jun phosphorylation in neurons of the peri-infarct area and Fas-L expression was reduced by FK506 treatment whereas ATF-2 expression was preserved.Cerebral ischemic damage to the brain was reduced by FK506. It was shown for the first time that neuroprotection by FK506 also included the suppression of the cerebral peptidyl-prolyl cis/trans isomerase activity of FKBP in vivo whereas the expression levels of FKBP12, 52 and 65 following ischemia changed slightly and FK506 treatment does not suppress the expression patterns. However, changes of FKBP enzymatic activity result in suppression of the stress cell body response in the peri-infarct area as observed by suppression of c-Jun phosphorylation and Fas-L expression.

JunB and Bcl-2 overexpression results in protection against cell death of nigral neurons following axotomy

Transection of the medial forebrain bundle is a well established approach to investigate neuronal cell body response in the derived neuronal populations of the substantia nigra pars compacta (SNC). This model of central axotomy leads in mouse within 50 days post transection to degeneration of up to 70% of the affected SNC neurons. A central component of the axotomy induced alterations leading to neuronal degeneration is the rapid induction, lasting expression and activation of the c-Jun transcription factor. However, the role of c-Jun in the process of neuronal degeneration is not fully understood. Since null mutations of c-Jun cause embryonic lethality, this study was designed to investigate the impact of two c-Jun modulating proteins on neuronal survival after axotomy in transgenic mice: JunB, a Jun family member affecting c-Jun expression, and Bcl-2, an antiapoptotic protooncogene interacting among others with the c-Jun N-terminal kinases. In JunB as well as in Bcl-2 transgenic mice the long term survival rate of transected SNC neurons was remarkably increased when compared to wildtype controls. These effects were obviously achieved by cellular modulations directly following axotomy: Whereas JunB overexpression attenuated c-Jun induction and simultaneously led to a higher phosphorylation rate of c-Jun in SNC neurons, Bcl-2 overexpression did not influence c-Jun expression, but resulted in a reduced phosphorylation state of c-Jun in transected SNC neurons. We therefore conclude that the early phosphorylation rate of c-Jun might play an important role for the long term fate of transected neurons.

Promoting axonal regeneration in the central nervous system by enhancing the cell body response to axotomy.

Neurons projecting into the peripheral nervous system (PNS) regenerate their axons after injury, in contrast to those confined to the central nervous system (CNS). Both neuronal and nonneuronal factors contribute to the lack of CNS regeneration. In this review we concentrate on the differential gene expression response to axotomy in PNS vs. CNS neurons. In general CNS neurons fail to up-regulate or sustain the expression of regeneration-associated proteins (RAGs), including trophic factors and their receptors. The presumed lack of trophic support of axotomized CNS neurons provided the rationale for the exogenous application of trophic factors, either to the lesion site or to the cell bodies. Here, we review our data on the application of trophic factors to rubrospinal and corticospinal neurons. Cell body treatment of axotomized rubrospinal neurons with brain-derived neurotrophic factor (BDNF) reversed atrophy, increased GAP-43 and Talpha-1 tubulin mRNA expression, and promoted axonal regeneration into peripheral nerve grafts. Importantly, BDNF cell body treatment was still effective in the chronic setting, i.e., when initiated 1 year after injury, but BDNF had no effect when applied to the chronic spinal cord injury site. The ability to promote regeneration in chronically injured neurons will hopefully contribute to the development of treatment strategies for chronic spinal injuries.

Central nervous system regeneration: mission impossible?

1. Attempts to induce clinical repair after central nervous system injury, such as spinal cord damage, are likely to involve several protocols because eliciting a regenerative response from an injured central neuron is a complex task. Future treatments, applied when a window of opportunity exists, address the requirements for regeneration. 2. Application of trophic support to the lesion site for axotomized neurons aims to initiate and maintain a cell body response conducive to axonal regrowth. 3. Surgical intervention may provide a bridge across the injury site that contains either Schwann cells or olfactory bulb ensheathing cells derived from the patient's own tissue. 4. The application of antibodies may block the inhibitory action of myelin-associated molecules and other glial elements. 5. Gene therapy may induce the correct cascade of guidance molecules to be released at appropriate times. 6. Physical rehabilitation may ensure that muscle wastage is reduced and encourages functional reconnection.

Two-Tiered Inhibition of Axon Regeneration at the Dorsal Root Entry Zone

Glial-derived inhibitory molecules and a weak cell-body response prevent sensory axon regeneration into the spinal cord after dorsal root injury. Neurotrophic factors, particularly neurotrophin-3 (NT-3), may increase the regenerative capacity of sensory neurons after dorsal rhizotomy, allowing regeneration across the dorsal root entry zone (DREZ). Intrathecal NT-3, delivered at the time of injury, promoted an upregulation of the growth-associated protein GAP-43 primarily in large-diameter sensory profiles (which did not occur after rhizotomy alone), as well as regeneration of cholera toxin B-labeled sensory axons across the DREZ and deep into the dorsal horn. However, delaying treatment for 1 week compromised regeneration: although axons still penetrated the DREZ, growth within white matter was qualitatively and quantitatively restricted. This was not associated with an impaired cell-body response (GAP-43 upregulation was equivalent for both immediate and delayed treatments), or with astrogliosis at the DREZ, which begins almost immediately after rhizotomy, but with the delayed appearance of mature ED1-expressing phagocytes in the dorsal white matter between 1 and 2 weeks after lesion, marking the beginning of myelin breakdown. After rhizotomy with immediate NT-3 treatment, regeneration continues beyond 2 weeks, but in the dorsal gray matter rather than in the degenerating dorsal columns. The ability of NT-3 to promote regeneration across the DREZ, but not after the beginning of degeneration after delayed treatment reveals a hierarchy of inhibitory influences: the astrogliotic, but not the degenerative barrier is surmountable by NT-3 treatment.

Detection of retrogradely transported WGA-HRP in axotomized adult hamster facial motoneurons occurs after initiation of the axon reaction.

We have previously shown that facial nerve transection at the stylomastoid foramen activates ribosomal RNA transcription in injured facial motoneurons (FMN) of the adult hamster within 30 minutes postoperative. The signal for the initiation of the nerve cell body response to injury in vertebrates is currently unknown. It has been hypothesized that the signal for initiating the injury response is dependent on retrograde transport, where the signal itself is either the loss of a repressor substance from the periphery or the loss of retrogradely transported target-derived factors. To examine if a retrograde transport-mediated signal would be sufficient to produce the rapid ribosomal effects observed in hamster FMN following injury, adult hamsters were subjected to right facial nerve axotomies, with the neuronal tracer wheat germ agglutinin horseradish peroxidase (WGA-HRP; M.W. 80,000) applied at the proximal stump of the transected nerve. At time points ranging from 0.5 to 24 hours postoperative (hpo), the animals were killed and brainstem sections containing bilateral facial nuclei processed for WGA-HRP label using the TMB method. The earliest time point at which WGA-HRP was detected in the axotomized facial nucleus occurred at 3 hpo. To eliminate molecular weight as a confounding factor, an additional retrograde transport study was performed using the smaller tracer, Fluoro-Gold (M.W. 532.59). Fluoro-Gold was not detected until well after the 3 hpo time point. Thus, it appears that initiation of the axon reaction in hamster FMN is likely to be independent of the retrograde transport properties of the injured neuron.

Cell Surface Trk Receptors Mediate NGF-Induced Survival While Internalized Receptors Regulate NGF-Induced Differentiation

Internalization and transport of a ligand-receptor complex are required to initiate cell body responses to target-derived neurotrophin. However, it is not known whether internalized receptors and cell surface receptors initiate the same signaling pathways and biological responses. Here we use a temperature-sensitive mutant of dynamin (G273D) to control the subcellular localization of activated NGF receptors (Trks). We show that dynamin function is required for ligand-dependent endocytosis of Trk receptors. In PC12 cells, nerve growth factor (NGF) stimulation promotes both survival and neuronal differentiation. These distinct biological responses to NGF are controlled by receptors signaling from different locations within the cell. Neuronal differentiation is promoted by catalytically active Trks within endosomes in the cell interior. In contrast, survival responses are initiated by activated receptors at the cell surface where they orchestrate prolonged activation of the kinase Akt. Thus, interactions between Trk receptor tyrosine kinases and intracellular signaling molecules are dictated both by phosphotyrosine motifs within the receptors and by the intracellular location of phosphorylated receptors.

Immunological Myelin Disruption Does Not Alter Expression of Regeneration-Associated Genes in Intact or Axotomized Rubrospinal Neurons

The inability of axotomized neurons to regenerate within the CNS has been partially attributed to a number of inhibitory factors associated with CNS myelin that are extrinsic to the severed neurons. However, some neurons are capable of limited regeneration after injury and this ability has been shown to correlate with the expression of certain regeneration-associated genes (RAGs) intrinsic to injured neurons. It has therefore been postulated that neutralization of inhibitory factors, as well as the induction of an appropriate neuronal cell body response, would facilitate improved regrowth of injured CNS axons. In previous studies we have shown that immunological removal of myelin from the spinal cord facilitates axonal regeneration by rubrospinal neurons, as indicated by retrograde transport of a fluorescent dye placed distal to the site of injury. Here, we investigated whether the immunological focal removal of spinal cord myelin, following a thoracic spinal cord injury, concomitantly stimulated an increase in the expression of RAGs in rubrospinal neurons. In situ hybridization for Tα-1 tubulin and GAP-43 at days 7, 14, and 21 revealed no significant increase in gene expression in rubrospinal neurons following immunological demyelination. The ability of various neuronal populations to sprout or slowly regrow without expressing the previously characterized cell body response is reviewed. We conclude that the recently demonstrated regeneration of rubrospinal tract, after immunologically directed spinal cord demyelination, is the result of either axonal sprouting or slow axonal regrowth without the increased expression of RAGs characteristic for fast axon regeneration.

Evolution of the Purkinje cell response to injury and regenerative potential during postnatal development of the rat cerebellum.

To understand the mechanisms leading to the progressive loss of intrinsic neuronal growth properties during central nervous system development, we have investigated the evolution of the response to injury and regenerative potential of immature Purkinje cells, axotomized at different postnatal ages from postnatal day (P)3 to P12. In adult rodents, these neurons are characterised by a weak cell body response to axotomy, which is associated with a remarkable resistance to injury and a poor regenerative capability. During the first postnatal week, Purkinje cells are strongly sensitive to injury and massively degenerate within a few days. Immature Purkinje cells react to neurite transection by a strong upregulation of c-Jun, accompanied by a moderate, but consistent, expression of the growth-associated protein (GAP)-43. In contrast, nicotinamide adenine dinucleotide monophosphate (NADPH)-diaphorase reactivity, which can be activated by adult Purkinje neurons, is not modified in their juvenile counterparts. The severed Purkinje axons show a vigorous regenerative sprouting both into the lesioned cerebellar environment and into embryonic neocortical tissue transplanted into the injury site. The typical adult features of the response to injury progressively develop during the second postnatal week, when the injured neurons acquire resistance, cell body changes become milder, the regenerative potential declines, and the severed axons undergo characteristic morphological modifications, including torpedoes and the hypertrophy of recurrent collateral branches. This complete reversal of the features and the outcome of the Purkinje cell reaction to axotomy likely results from the profound changes that occur in the maturing Purkinje cells and/or in their microenvironment during this phase of cerebellar development.

The immunophilin ligand FK506, but not GPI-1046, protects against neuronal death and inhibits c-Jun expression in the substantia nigra pars compacta following transection of the rat medial forebrain bundle

The immunophilin ligand FK506 (Tacrolimus) is used for prevention of graft rejection following organ transplantation. FK506 is a high-affinity ligand for FK506-binding proteins, an immunophilin subgroup of peptidyl-prolyl- cis/ trans-rotamases abundant in the mammalian brain. Here, we demonstrate that FK506 is a potent survival factor that prevents neuronal cell death following axotomy of central intrinsic neurons. Administration of FK506 (2 mg/kg, s.c., per day for two days pre-axotomy and for up to eight days post-axotomy) effectively delayed and reduced the death of axotomized neurons in the substantia nigra pars compacta following transection of the medial forebrain bundle. In saline-treated controls, 75%, 89% and 92% of nigral neurons died after 25, 50 and 60 days post-axotomy, respectively. In contrast, application of FK506 resulted in survival of 46%, 44% and 28% of the axotomized nigral neurons, and the majority of these surviving neurons showed continuous expression of tyrosine hydroxylase, the pacemaker enzyme for dopamine synthesis. Moreover, FK506 significantly reduced the expression of the inducible transcription factor c-Jun and its N-terminal phosphorylation and prevented the axotomy-induced suppression of the constitutive transcription factor ATF-2 in neurons of the substantia nigra and mammillary body. The latter is also axotomized by the coincident transection of the mammillothalamic tract, but the mammillary neurons survive the axotomy. In contradistinction to FK506, the non-immunosuppressive FK506-binding protein ligand GPI-1046 (25 or 12.5 mg/kg, applied once or twice per day for two days pre-axotomy and for eight days post-axotomy) was completely ineffective for all these parameters investigated. Finally, FK506, but not GPI-1046, impressively accelerated the recovery from surgery. Our data provide the first evidence that FK506 acts as a neuroprotective molecule that rescues axotomized otherwise degenerating central intrinsic neurons in the adult mammalian brain by mechanisms that interfere with the transcriptional program of the axotomy-induced cell body response, such as activating transcription factor-2 suppression and c-Jun expression and phosphorylation.