RESUMEN
The polyketides FK506 (tacrolimus) and FK520 (ascomycin) are potent immunosuppressants that function by inhibiting calcineurin phosphatase through formation of an FKBP12-FK506/520-calcineurin ternary complex. They also have calcineurin-independent neuroregenerative properties in cell culture and animal models of nervous system disorders. Based on the crystal structure of the FKBP12-FK506-calcineurin complex, we deduced that the 13- and 15-methoxy groups of FK506 or FK520 are important for inhibition of calcineurin phosphatase but not for binding to FKBP12. By genetic modification of the FK520 gene cluster, we generated 13- and 15-desmethoxy analogs of FK520 that contain hydrogen, methyl, or ethyl instead of methoxy at one or both of these positions. These analogs bind FKBP12 tightly, have decreased calcineurin phosphatase inhibition and immunosuppressive properties, and enhance neurite outgrowth in cell cultures. A representative compound was also shown to accelerate nerve regeneration and functional recovery in the rat sciatic nerve crush model.
Asunto(s)
Inmunosupresores/farmacología , Regeneración Nerviosa/efectos de los fármacos , Streptomyces/genética , Tacrolimus/farmacología , Acetiltransferasas/genética , Acetiltransferasas/metabolismo , Animales , Calcineurina/metabolismo , Línea Celular , Vectores Genéticos , Hipocampo/citología , Hipocampo/efectos de los fármacos , Humanos , Compresión Nerviosa , Neuritas/efectos de los fármacos , Unión Proteica , Ingeniería de Proteínas , Ratas , Proteínas Recombinantes/farmacología , Nervio Ciático/efectos de los fármacos , Nervio Ciático/patología , Streptomyces/metabolismo , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Tacrolimus/análogos & derivadosRESUMEN
We compared the effects of FK506 administration on regeneration and reinnervation after sciatic nerve resection and repair with an autologous graft or with a silicone tube leaving a 6-mm gap in the mouse. Functional reinnervation was assessed by noninvasive methods to determine recovery of motor, sensory, and sweating functions in the hindpaw over 4 months after operation. Morphometric analysis of the regenerated nerves was performed at the end of follow-up. The nerve graft allowed for faster and higher levels of reinnervation in the four functions tested than silicone tube repair. Treatment with FK506 (for the first 9 weeks only) resulted in a slight, although not significant, improvement of the onset of reinnervation and of the maximal degree of recovery achieved after autografting. The recovery of pain sensibility and of the compound nerve action potentials in the digital nerves, which directly depend on axonal regeneration, showed better progression with FK506 than reinnervation of muscles and sweat glands, which require reestablishment of synaptic contacts with target cells. The myelinated fibers in the regenerated nerve showed a more mature appearance in the FK506-treated rats. However, FK506 showed a marginal effect in situations in which regeneration was limited, as in a silicone tube bridging a 6-mm gap in the mouse sciatic nerve. In conclusion, treatment with FK506 improved the rate of functional recovery after nerve resection and autograft repair.
Asunto(s)
Inmunosupresores/farmacología , Regeneración Nerviosa/efectos de los fármacos , Nervio Ciático/fisiología , Nervio Ciático/trasplante , Tacrolimus/farmacología , Potenciales de Acción/fisiología , Animales , Femenino , Ratones , Ratones Endogámicos , Fibras Nerviosas Mielínicas/fisiología , Regeneración Nerviosa/fisiología , Conducción Nerviosa/fisiología , Umbral del Dolor/fisiología , Recuperación de la Función/efectos de los fármacos , Recuperación de la Función/fisiología , Nervio Ciático/cirugía , Siliconas , Trasplante AutólogoRESUMEN
Neuroimmunophilin ligands are a class of compounds that hold great promise for the treatment of nerve injuries and neurological disease. In contrast to neurotrophins (e.g., nerve growth factor), these compounds readily cross the blood-brain barrier, being orally effective in a variety of animal models of ischaemia, traumatic nerve injury and human neurodegenerative disorders. A further distinction is that neuroimmunophilin ligands act via unique receptors that are unrelated to the classical neurotrophic receptors (e.g., trk), making it unlikely that clinical trials will encounter the same difficulties found with the neurotrophins. Another advantage is that two neuroimmunophilin ligands (cyclosporin A and FK-506) have already been used in humans (as immunosuppressant drugs). Whereas both cyclosporin A and FK-506 demonstrate neuroprotective actions, only FK-506 and its derivatives have been clearly shown to exhibit significant neuroregenerative activity. Accordingly, the neuroprotective and neuroregenerative properties seem to arise via different mechanisms. Furthermore, the neuroregenerative property does not involve calcineurin inhibition (essential for immunosuppression). This is important since most of the limiting side effects produced by these drugs arise via calcineurin inhibition. A major breakthrough for the development of this class of compounds for the treatment of human neurological disorders was the ability to separate the neuroregenerative property of FK-506 from its immunosuppressant action via the development of non-immunosuppressant (non-calcineurin inhibiting) derivatives. Further studies revealed that different receptor subtypes, or FK-506-binding proteins (FKBPs), mediate immunosuppression and nerve regeneration (FKBP-12 and FKBP-52, respectively, the latter being a component of steroid receptor complexes). Thus, steroid receptor chaperone proteins represent novel targets for future drug development of novel classes of compounds for the treatment of a variety of human neurological disorders, including traumatic injury (e.g., peripheral nerve and spinal cord), chemical exposure (e.g., vinca alkaloids, Taxol) and neurodegenerative disease (e.g. , diabetic neuropathy and Parkinson's disease).
Asunto(s)
Ciclosporina/uso terapéutico , Inmunosupresores/uso terapéutico , Regeneración Nerviosa/fisiología , Enfermedades del Sistema Nervioso/tratamiento farmacológico , Proteína 1A de Unión a Tacrolimus/fisiología , Tacrolimus/uso terapéutico , Animales , Lesiones Encefálicas/tratamiento farmacológico , Síndrome del Túnel Carpiano/tratamiento farmacológico , Ciclosporina/farmacología , Humanos , Inmunofilinas/farmacología , Inmunofilinas/uso terapéutico , Inmunosupresores/farmacología , Ligandos , Regeneración Nerviosa/efectos de los fármacos , Traumatismos de la Médula Espinal/tratamiento farmacológico , Tacrolimus/química , Tacrolimus/farmacología , Proteína 1A de Unión a Tacrolimus/efectos de los fármacosRESUMEN
In summary, FKBP-12 does not mediate the neurite outgrowth-promoting properties of neuroimmunophilin ligands (e.g., FK506). Instead, the neurotrophic properties of neuroimmunophilin ligands (FK506) and steroid hormones are mediated by disruption of steroid-receptor complexes. It remains unclear which component mediates neurite outgrowth, although the most likely candidates are FKBP-52, hsp-90, and p23 [42]. Regardless of the underlying mechanism involved, the FKBP-52 antibody data reveal that it should be possible to design, based on the structure of FK506, non-FKBP-12-binding (nonimmunosuppressant) compounds selective for FKBP-52 and test these new libraries for their ability to augment nerve regeneration. It may also be possible to exploit the structure of geldanamycin to develop a new class of hsp-90-binding compounds for use in nerve regeneration.
Asunto(s)
Inmunofilinas/inmunología , Regeneración Nerviosa/inmunología , Tacrolimus/farmacología , Animales , Anticuerpos/inmunología , Benzoquinonas , Humanos , Inmunosupresores/farmacología , Lactamas Macrocíclicas , Ratones , Ratones Noqueados , Molibdeno/farmacología , Neuritas/efectos de los fármacos , Neuritas/fisiología , Quinonas/farmacología , Esteroides/farmacología , Proteínas de Unión a TacrolimusRESUMEN
We examined whether the nerve regenerative property of FK506 exhibits a 'window-of-opportunity' corresponding to the time of injury for maximal efficacy in the sciatic nerve crush model. FK506 (5 mg/kg, s.c.) was administered over the 18-day period of study according to three dosage regiments: delayed (days 9-17), discontinuous (days 0-8) and continuous (days 0-17) administrations. Quantitation of axonal calibers and the extend of myelination in the soleus nerve at 18 days demonstrated that both delayed and discontinuous administrations were equally effective, arguing against a 'window-of-opportunity' for FK506 nerve regenerative effect. However, both protocols were less effective than continuous administration indicating that the compound needs to be given during the entire regenerative period to elicit maximal efficacy.
Asunto(s)
Compresión Nerviosa/métodos , Regeneración Nerviosa/efectos de los fármacos , Nervio Ciático/fisiología , Tacrolimus/administración & dosificación , Animales , Axones/efectos de los fármacos , Axones/fisiología , Axones/ultraestructura , Inyecciones Subcutáneas , Microscopía Electrónica , Fibras Nerviosas Mielínicas/efectos de los fármacos , Fibras Nerviosas Mielínicas/fisiología , Fibras Nerviosas Mielínicas/ultraestructura , Ratas , Ratas Sprague-Dawley , Nervio Ciático/efectos de los fármacos , Nervio Ciático/ultraestructura , Factores de TiempoRESUMEN
The neurotrophic property of the immunosuppressant drug FK506 (tacrolimus) is believed to depend on the 12-kDa FK506-binding protein (FKBP-12). Here, we show that FK506 maintains its neurotrophic activity in primary hippocampal cell cultures from FKBP-12 knockout mice. In human neuroblastoma SH-SY5Y cells, the neurotrophic action of FK506 (10 pM to 10 nM) is completely prevented by the addition of a monoclonal antibody (50-100 nM) to the immunophilin FKBP-52 (also known as FKBP-59 or heat shock protein 56), a component of mature steroid receptor complexes. By itself, the FKBP-52 antibody is also neurotrophic. The neurotrophic activity of dexamethasone (50 nM) is potentiated by FK506, whereas that of beta-estradiol (50 nM) is not altered, suggesting a common mechanisms of action. Geldanamycin (which disrupts mature steroid receptor complexes) is also neurotrophic (0.1-10 nM), whereas it reduces the neurotrophic activity of FK506 and steroid hormones (dexamethasone and beta-estradiol). Conversely, 20 mM molybdate (which prevents the disruption of mature steroid receptor complexes) decreases the neurotrophic activity of FK506, FKBP-52 antibody, dexamethasone, and beta-estradiol. In rats, FK506 (10 mg/kg s.c.) augments the regenerative response of regenerating motor and sensory neurons to nerve injury as shown by its ability to increase the axotomy-induced induction of c-jun expression. A model is proposed to account for the neurotrophic action of both neuroimmunophilin ligands (FK506) and steroid hormones. Components of steroid receptor complexes represent novel targets for the rational design of new neurotrophic drugs.
Asunto(s)
Hipocampo/fisiología , Inmunofilinas/fisiología , Factores de Crecimiento Nervioso/farmacología , Neuronas/fisiología , Fármacos Neuroprotectores/farmacología , Nervio Ciático/fisiología , Tacrolimus/farmacología , Animales , Anticuerpos/farmacología , Benzoquinonas , Células Cultivadas , Inhibidores de Cisteína Proteinasa/farmacología , Dexametasona/farmacología , Embrión de Mamíferos , Estradiol/farmacología , Humanos , Inmunofilinas/deficiencia , Inmunofilinas/inmunología , Lactamas Macrocíclicas , Ratones , Ratones Noqueados , Molibdeno/farmacología , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/fisiología , Regeneración Nerviosa/efectos de los fármacos , Neuritas/efectos de los fármacos , Neuritas/fisiología , Neuroblastoma , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas Aferentes/efectos de los fármacos , Neuronas Aferentes/fisiología , Proteínas Proto-Oncogénicas c-jun/genética , Quinonas/farmacología , Ratas , Ratas Sprague-Dawley , Nervio Ciático/efectos de los fármacos , Nervio Ciático/lesiones , Proteínas de Unión a Tacrolimus , Células Tumorales CultivadasRESUMEN
The authors examined the ability of FK506 to accelerate axonal regeneration of rat spinal cord axons in a peripheral nerve (PN) graft. Predegenerated autografts were produced by transecting the left tibial nerve 1 week prior to spinal cord implantation into the lumbar (L-3-L-4) spinal cord. Rats were given daily injections of either FK506 (5 mg/kg, subcutaneous) or vehicle for 21 days. The PN grafts from FK506-treated rats contained larger sized regenerating axons compared with vehicle-treated controls, and mean axonal areas increased by 25% at 7.5 mm along the PN graft. Fluoro-Gold retrograde labeling confirmed that the regenerating axons originated from the central nervous system. Unexpectedly, the majority (>50%) of neurons in the red nucleus were retrogradely labeled in the FK506-treated animals only. The results indicate that FK506 not only accelerates the elongation of spinal cord axons but also promotes regeneration of rubrospinal neurons.
Asunto(s)
Axones/efectos de los fármacos , Inmunosupresores/farmacología , Degeneración Nerviosa/patología , Regeneración Nerviosa/efectos de los fármacos , Nervios Periféricos/trasplante , Traumatismos de la Médula Espinal/patología , Tacrolimus/farmacología , Animales , Axones/patología , Masculino , Microscopía Electrónica , Vías Nerviosas/efectos de los fármacos , Vías Nerviosas/patología , Neuronas/efectos de los fármacos , Neuronas/patología , Ratas , Ratas Sprague-Dawley , Núcleo Rojo/efectos de los fármacos , Núcleo Rojo/patología , Médula Espinal/efectos de los fármacos , Médula Espinal/patologíaRESUMEN
FK506, an immunosuppressant drug used to prevent allograft rejection in organ transplantations, accelerates functional recovery and nerve regeneration in the rat sciatic nerve crush model. While the mechanism by which FK506 increases regeneration is unknown, in contrast to immunosuppression, it does not involve calcineurin inhibition. Using the reverse-transcriptase polymerase chain reaction (RT-PCR) technique and a digoxigenin-labeled probe, we show that subcutaneous injections of FK506 (10 mg/kg/day) markedly increases the level of axotomy-induced growth-associated protein (GAP-43) mRNA in dorsal root ganglion (DRG) neurons. Quantitation of DRG neurons revealed that FK506 produced a 33% increase in the numbers of neurons exhibiting intense staining. Increased synthesis of GAP-43 may play a role in FK506's ability to speed nerve regeneration.
Asunto(s)
Proteína GAP-43/genética , Inmunosupresores/administración & dosificación , Neuronas Aferentes/metabolismo , ARN Mensajero/metabolismo , Tacrolimus/administración & dosificación , Animales , Axotomía , Proteína GAP-43/efectos de los fármacos , Proteína GAP-43/metabolismo , Inyecciones Subcutáneas , Neuronas Aferentes/efectos de los fármacos , Neuronas Aferentes/fisiología , ARN Mensajero/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Nervio Ciático/fisiologíaRESUMEN
We recently showed that s.c. injections of a nonimmunosuppressant FK506 binding protein-12 (FKBP-12) ligand (V-10,367) accelerates nerve regeneration in the rat sciatic nerve crush model. Here we examined the oral efficacy of this compound for speeding nerve regeneration. Rats receiving V-10,367 (5, 15 or 50 mg/kg/day) by oral gavage all demonstrated an increase in nerve regeneration compared to vehicle-treated controls. Functional recovery was observed earliest and axonal calibers of regenerating axons in the soleus nerve were largest in the 15 mg/kg group, mean axonal areas being increased by 66% compared to controls. Orally active nonimmunosuppressant FKBP-12 ligands may be useful for the treatment of human peripheral nerve disorders.
Asunto(s)
Proteínas Portadoras/farmacología , Proteínas de Unión al ADN/farmacología , Proteínas de Choque Térmico/farmacología , Regeneración Nerviosa/efectos de los fármacos , Administración Oral , Análisis de Varianza , Animales , Inmunosupresores/farmacología , Inyecciones Subcutáneas , Ligandos , Masculino , Microscopía Electrónica , Compresión Nerviosa , Ratas , Ratas Sprague-Dawley , Nervio Ciático/lesiones , Proteínas de Unión a TacrolimusRESUMEN
The immunosuppressant drugs FK506 and cyclosporin A inhibit T-cell proliferation via a common mechanism: calcineurin inhibition following binding to their respective binding proteins, the peptidyl prolyl isomerases FKBP-12 and cyclophilin A. In contrast, FK506, but not cyclosporin A, accelerates nerve regeneration. In the present study, we show that the potent FKBP-12 inhibitor V-10,367, which lacks the structural components of FK506 required for calcineurin inhibition, increases neurite outgrowth in SH-SY5Y neuroblastoma cells and speeds nerve regeneration in the rat sciatic nerve crush model. In SH-SY5Y cells, V-10,367 increased the lengths of neurite processes in a concentration-dependent (between 1 and 10 nM) fashion over time (up to 168 h). Daily subcutaneous injections of V-10,367 accelerated the onset of clinical signs of functional recovery in the hind feet compared to vehicle-treated control animals. Interdigit distances (between the first and fifth digits) measured on foot prints obtained during walking showed an increase in toe spread in V-10,367-treated rats compared to vehicle-treated controls. Electron microscopy demonstrated larger regenerating axons distal to the crush site in the sciatic nerve from V-10,367-treated rats. Quantitation of axonal areas in the soleus nerve revealed a shift to larger axonal calibers in V-10,367-treated rats (400 or 200 mg/kg/day); mean axonal areas were increased by 52 and 59%, respectively, compared to vehicle-treated controls. FKBP-12 ligands lacking calcineurin inhibitory activity represent a new class of potential drugs for the treatment of human peripheral nerve disorders.
Asunto(s)
Proteínas Portadoras/antagonistas & inhibidores , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Choque Térmico/antagonistas & inhibidores , Regeneración Nerviosa/efectos de los fármacos , Neuritas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Piridinas/farmacología , Nervio Ciático/fisiología , Tacrolimus/farmacología , Animales , Inhibidores de la Calcineurina , Proteínas Portadoras/fisiología , Proteínas de Unión al ADN/fisiología , Evaluación Preclínica de Medicamentos , Proteínas de Choque Térmico/fisiología , Humanos , Inyecciones Subcutáneas , Locomoción , Masculino , Estructura Molecular , Compresión Nerviosa , Neuritas/ultraestructura , Neuroblastoma/patología , Fármacos Neuroprotectores/administración & dosificación , Fármacos Neuroprotectores/farmacocinética , Fármacos Neuroprotectores/uso terapéutico , Piridinas/administración & dosificación , Piridinas/farmacocinética , Piridinas/uso terapéutico , Ratas , Ratas Sprague-Dawley , Nervio Ciático/efectos de los fármacos , Nervio Ciático/lesiones , Estimulación Química , Relación Estructura-Actividad , Tacrolimus/química , Proteínas de Unión a Tacrolimus , Células Tumorales CultivadasRESUMEN
The new immunosuppressant drug FK506 (Tacrolimus) increases the rate of nerve regeneration in vivo (Gold et al., 1994; Gold et al., 1995). In the present study, we have examined the dose-dependence of FK506's ability to enhance nerve regeneration. In the first set of experiments, rats received daily s.c. injections of FK506 (2 mg/kg, 5 mg/kg or 10 mg/kg) for 18 days after a sciatic nerve crush injury. Signs of functional recovery in the hind feet appeared earlier than in saline-treated control rats at all three FK506 dosage; recovery was maximally accelerated in the 5-mg/kg group. Light microscopy at 18 days after nerve crush revealed more regenerating myelinated fibers in FK506-treated rats than in controls; this was most apparent in the 5-mg/kg group. Morphometric analysis of axonal areas in the soleus nerve confirmed that axonal calibers were maximally increased in the 5-mg/kg group. In the second set of experiments, the rate of axonal regeneration was determined by radiolabeling the L5 dorsal root ganglion. Regeneration rate for sensory axons was maximally increased (by 34%) in the 5-mg/kg group. In contrast, cyclosporin A (10 or 50 mg/kg; dosages were selected on the basis of the 1/10 lower potency of cyclosporin A) did not significantly alter the rate of axonal regeneration. Cyclosporin A (50 mg/kg) also failed to increase functional recovery or axonal calibers in the soleus nerve. Because the two drugs share a common mechanism for producing immunosuppression (i.e., calcineurin inhibition), these results indicate that FK506's nerve regenerative property involves a distinct, calcineurin-independent mechanism.
Asunto(s)
Axones/efectos de los fármacos , Ciclosporina/farmacología , Regeneración Nerviosa/efectos de los fármacos , Nervio Ciático/efectos de los fármacos , Tacrolimus/farmacología , Animales , Axones/fisiología , Axones/ultraestructura , Ciclosporina/administración & dosificación , Relación Dosis-Respuesta a Droga , Masculino , Microscopía Electrónica , Ratas , Ratas Sprague-Dawley , Nervio Ciático/fisiología , Nervio Ciático/ultraestructura , Tacrolimus/administración & dosificaciónRESUMEN
While it is well established that nerve growth factor is growth promoting for sensory neurons in culture, it is unclear whether it serves such a function in vivo. In fact, our previous studies led to the hypothesis that nerve growth factor could actually impair axonal regeneration by reducing the neuronal cell body response to injury. In the present study, the consequence of continuous intrathecal infusion of nerve growth factor on regeneration of sensory neurons was examined in rats given a bilateral sciatic nerve crush. Rats received nerve growth factor (125 ng/h) as a continuous infusion into the subarachnoid space of the lumbar spinal cord via an osmotic minipump (Alzet); controls received cytochrome C. At seven or 10 days, the pump was removed and L4 or L5 dorsal root ganglion exposed and injected with 50 microCi of (3H)leucine. Animals were killed 24 h later, the sciatic nerves removed, cut into 3 mm segments and the radioactivity in each segment determined by liquid scintillation spectrophotometry. Maximal regeneration distances (determined from the front of the resultant transport curves) were similarly reduced (by approximately 6 mm) in nerve growth factor-infused compared to cytochrome C-infused rats. Thus, regeneration rates (determined between eight and 11 days) were unaltered by nerve growth factor infusion; regeneration rates from cytochrome C-infused and nerve growth factor-infused animals were 2.8 mm/day and 3.1 mm/day, respectively. However, nerve growth factor significantly (P < 0.005) increased the delay to onset for regeneration by two days. Taken together, the present study demonstrates that nerve growth factor delays the onset of regeneration without affecting the rate of regeneration. The results implicate the involvement of at least two signals in the regulation of axonal regeneration in dorsal root ganglion neurons. It is suggested that the loss of nerve growth factor serves as an early, induction signal regulating the onset of regeneration and that a second, unidentified signal independently serves to maintain regeneration.
Asunto(s)
Axones/efectos de los fármacos , Factores de Crecimiento Nervioso/farmacología , Regeneración Nerviosa/efectos de los fármacos , Neuronas Aferentes/efectos de los fármacos , Animales , Ganglios Espinales/citología , Ganglios Espinales/efectos de los fármacos , Inyecciones Espinales , Masculino , Compresión Nerviosa , Factores de Crecimiento Nervioso/administración & dosificación , Ratas , Ratas Sprague-Dawley , Nervio Ciático/efectos de los fármacos , Nervio Ciático/fisiología , Nervio Ciático/ultraestructuraRESUMEN
To develop unilateral, chronically elevated intraocular pressure in rats, episcleral veins were injected with hypertonic saline and the intraocular pressure was monitored with a Tono-Pen XL tonometer. Histologic analyses of eyes with differing degrees and durations of intraocular pressure elevation were performed to ascertain the effects of these pressures on the optic nerve. Out of 20 consecutive animals, nine had elevations of intraocular pressure following a single injection, while subsequent injections raised intraocular pressure in seven others. One eye became hypotonous. In the remaining animals, subsequent injections sufficient to raise intraocular pressure were deliberately withheld, to determine the possible direct effects of injections on the optic nerve. Mean sustained pressure elevations ranged from 7 to 28 mm Hg and the retinal vasculature remained perfused in all eyes. Optic nerve cross sections from eyes without intraocular pressure elevation appeared identical to those from uninjected eyes, while nerves from eyes with the greatest intraocular pressure rise demonstrated axonal damage that involved 100% of the neural area. Eyes with either less severe pressure elevations or shorter durations showed partial damage, ranging from 0.5% to 10.4% of the neurla area. In 70% of these nerves, damage was concentrated in the superior temporal region. Within the optic nerve head, often associated with astrocytes, axons contained abnormal accumulations of membrane-bound vesicles and mitochondria. The anterior chamber angles showed sclerosis of the trabecular meshwork with anterior synechiae, but Schlemm's canal, collector channels and aqueous veins appeared patent. Unilateral sclerosis of the trabecular meshwork produces sustained elevation of intraocular pressure in rats with optic nerve damage that in many ways resembles that seen in human glaucoma. Understanding the mechanism of nerve damage in this model may provide new insights into the pathogenesis of human glaucoma.
Asunto(s)
Modelos Biológicos , Hipertensión Ocular/complicaciones , Enfermedades del Nervio Óptico/etiología , Animales , Masculino , Hipertensión Ocular/inducido químicamente , Hipertensión Ocular/fisiopatología , Enfermedades del Nervio Óptico/patología , Ratas , Cloruro de Sodio/efectos adversos , Malla Trabecular/patologíaRESUMEN
FK506 is a new FDA-approved immunosuppressant used for prevention of allograft rejection in, for example, liver and kidney transplantations. FK506 is inactive by itself and requires binding to an FK506 binding protein-12 (FKBP-12), or immunophilin, for activation. In this regard, FK506 is analogous to cyclosporin A, which must bind to its immunophilin (cyclophilin A) to display activity. This FK506-FKBP complex inhibits the activity of the serine/threonine protein phosphatase 2B (calcineurin), the basis for the immunosuppressant action of FK506. The discovery that immunophilins are also present in the nervous system introduces a new level of complexity in the regulation of neuronal function. Two important calcineurin targets in brain are the growth-associated protein GAP-43 and nitric oxide (NO) synthase (NOS). This review focuses on studies showing that systemic administration of FK506 dose-dependently speeds nerve regeneration and functional recovery in rats following a sciatic-nerve crush injury. The effect appears to result from an increased rate of axonal regeneration. The nerve regenerative property of this class of agents is separate from their immunosuppressant action because FK506-related compounds that bind to FKBP-12 but do not inhibit calcineurin are also able to increase nerve regeneration. Thus, FK506's ability to increase nerve regeneration arises via a calcineurin-independent mechanism (i.e., one not involving an increase in GAP-43 phosphorylation). Possible mechanisms of action are discussed in relation to known actions of FKBPs: the interaction of FKBP-12 with two Ca2+ release-channels (the ryanodine and inositol 1,4,5-triphosphate receptors) which is disrupted by FK506, thereby increasing Ca2+ flux; the type 1 receptor for the transforming growth factor-beta (TGF-beta 1), which stimulates nerve growth factor (NGF) synthesis by glial cells, and is a natural ligand for FKBP-12; and the immunophilin FKBP-52/FKBP-59, which has also been identified as a heat-shock protein (HSP-56) and is a component of the nontransformed glucocorticoid receptor. Taken together, studies of FK506 indicate broad functional roles for the immunophilins in the nervous system. Both calcineurin-dependent (e.g., neuroprotection via reduced NO formation) and calcineurin-independent mechanisms (i.e., nerve regeneration) need to be invoked to explain the many different neuronal effects of FK506. This suggests that multiple immunophilins mediate FK506's neuronal effects. Novel, nonimmunosuppressant ligands for FKBPs may represent important new drugs for the treatment of a variety of neurological disorders.
Asunto(s)
Inmunosupresores/uso terapéutico , Regeneración Nerviosa/efectos de los fármacos , Tacrolimus/uso terapéutico , Animales , Axones/fisiología , Inhibidores de la Calcineurina , Canales de Calcio/fisiología , Proteínas Portadoras/metabolismo , Células Cultivadas , Proteínas de Unión al ADN/metabolismo , Aprobación de Drogas , Proteínas de Choque Térmico/metabolismo , Humanos , Inmunosupresores/metabolismo , Compresión Nerviosa , Nervio Ciático/lesiones , Tacrolimus/metabolismo , Proteínas de Unión a TacrolimusRESUMEN
The axonal regenerative properties of the new immunosuppressant drug FK506 (tacrolimus) are further explored in this continuing study. In an initial report (Gold et al., 1994a), we described the ability of FK506 to reduce the time until return of function in the hind feet of rats following a sciatic nerve crush. In the present study, we examined the morphological correlate underlying this enhancement of functional recovery. In rats receiving daily subcutaneous injections of FK506 (1.0 mg/kg) for 18 d following a sciatic nerve crush the regenerating axons appeared larger in size compared to saline-injected control animals. Morphometric analysis of axonal calibers in the soleus nerve demonstrated that mean axonal areas for the largest 30% of axons were increased over axotomized control values by 93% in the FK506-treated animals. Next, the rate of axonal regeneration was determined by radiolabeling the L5 dorsal root ganglion (DRG) at 9 and 14 d following axotomy. Regression analysis of the outgrowth distances for sensory axons between 10 and 15 d revealed a 16% increase in regeneration rate. Electron microscopy of intramuscular nerve branches in the interosseus muscles confirmed that the axons in the FK506-treated animals were further advanced toward their targets; in some instances, axons were shown to reinnervate muscle spindles. The results are discussed in terms of the known ability of FK506 to inhibit the activity protein phosphatase 2B (calcineurin).
Asunto(s)
Axones/efectos de los fármacos , Inmunosupresores/farmacología , Regeneración Nerviosa/efectos de los fármacos , Nervio Ciático/efectos de los fármacos , Tacrolimus/farmacología , Animales , Axones/fisiología , Masculino , Neuronas Aferentes/efectos de los fármacos , Neuronas Aferentes/fisiología , Ratas , Ratas Sprague-Dawley , Nervio Ciático/citología , Factores de TiempoRESUMEN
We examined the axotomy-induced expression of the immediate-early gene (proto-oncogene) c-jun in the Ola mouse mutant (which exhibits a dramatic delay in Wallerian degeneration) using immunocytochemistry to c-JUN (the protein product of the protooncogene c-jun). c-JUN-like protein immunoreactivity was present in a similar proportion (ca. 60%) of L4 dorsal root ganglion (DRG) neuronal cell bodies from normal (C57/6J/BL) and Ola mice at 1 week following a sciatic nerve crush (axotomy). In normal mice, the intensity and extent of staining declined at 3 weeks, correlating with regeneration. In contrast, Ola mice exhibited a marked reduction (by 77%) in the extent of staining at 2 weeks. At 3 weeks (coinciding to the onset of extensive axonal degeneration in this mutant), staining levels were increased to 1 week levels. Taken together, these findings suggest that multiple signals (both independent and dependent on axonal degeneration) regulate c-jun expression in DRG neuronal cell bodies.
Asunto(s)
Axones/fisiología , Ganglios Espinales/metabolismo , Regeneración Nerviosa , Neuronas Aferentes/metabolismo , Proteínas Proto-Oncogénicas c-jun/biosíntesis , Nervio Ciático/fisiología , Animales , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes Neurológicos , Compresión Nerviosa , Proteínas Proto-Oncogénicas c-jun/análisis , Valores de Referencia , Especificidad de la EspecieRESUMEN
Regeneration of peripheral nerve fibers over long distances often requires extended periods of convalescence. Loss to society can be measured in terms of increased health care costs, decreased productivity and, in the case of job-related injuries, larger workers' compensation claims. The availability of drugs to increase axonal regeneration would be beneficial not only to patients but also to society in general by decreasing these costs. In the present paper, we present our initial studies on the regenerative effects of the new immunosuppressive agent FK506. Rats given a sciatic nerve crush (axotomy) received daily subcutaneous injections of FK506 (1.0 mg/kg); axotomized control animals received saline. Clinical signs of recovery in the hind feet were manifested two days earlier in FK506-treated than in saline-treated animals; movement in the toes, and walking on the hind feet and toes were observed at 16 and 17 days, respectively, in saline-treated rats and at 14 and 15 days, respectively, in FK506-treated rats. Measurement of interdigit distances in the hind feet at 18 days following axotomy showed a return toward normal position of the toes (increased interdigit distances) during walking in FK506-treated rats. Light and electron microscopy performed at 18 days following axotomy confirmed the clinical appearance of increased functional recovery in FK506-treated rats. Distal to the crush site, the sciatic nerve and its terminal branches from FK506-treated animals contained more myelinated fibers compared to saline-treated animals; in the soleus nerve, the numbers of myelinated axons was increased 2.75-fold. Taken together, the present results suggest that FK506 enhances recovery of function in the rat by increasing the rate of axonal regeneration following a sciatic nerve crush.
RESUMEN
Axonal regeneration over long distances is dependent upon events occurring both in the distal stump and in the neuronal cell body. Little is known concerning how events in the distal stump influence the cell body response to injury, or the axon reaction. In the present study, we examined this relationship for one component of the axon reaction (i.e. aberrant neurofilament (NF) phosphorylation) in the C57BL/Ola (Ola) mouse mutant, a model which exhibits delayed Wallerian degeneration (up to 3 weeks) and retarded regeneration of sensory neurons. Non-axotomized normal (C57/6J/BL) and Ola mice demonstrated modest immunostaining to phosphorylated NF (pNF) epitopes (using monoclonal antibody 06-17) in some (11%) L4 dorsal root ganglion (DRG) neuronal cell bodies. In normal mice, modest to intense immunoreactivity was present in 43% of DRG neurons at 1 week following a sciatic nerve crush (axotomy). The intensity and extent of staining declined with reinnervation, being reduced slightly at 2 weeks and more notably by 3 weeks following axotomy. In Ola mice, the intensity and extent (43%) of staining were not different from normal axotomized mice at 1 week following axotomy. However, the intensity was less and the extent of staining reduced by 28% at 2 weeks following axotomy. By 3 weeks, staining levels were again increased, being similar to that observed in Ola and normal mice at 1 week following axotomy.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Neurofibrillas/metabolismo , Neuronas Aferentes/metabolismo , Transducción de Señal/fisiología , Degeneración Walleriana/fisiología , Animales , Axones/fisiología , Epítopos , Técnicas para Inmunoenzimas , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes Neurológicos , FosforilaciónRESUMEN
We examined the regulation by nerve growth factor (NGF) of the immediate-early gene (proto-oncogene) c-jun in adult dorsal root ganglion (DRG) neurons using immunocytochemistry to c-JUN (the protein product of the proto-oncogene c-jun). Following a sciatic nerve crush, the injury-induced increase in c-JUN-like immunostaining was reduced in DRG neurons by continuous intrathecal infusion of NGF for one week. Conversely, in intact DRG neurons (i.e., without Wallerian degeneration), c-JUN-like immunoreactivity was markedly increased following four weeks of daily NGF antiserum injections (to remove target tissue-derived NGF) into the hindfoot. Taken together, these findings indicate that nerve transection (axotomy) results in a loss of target tissue-derived NGF leading to induction of the transcription factor c-jun which may play a role in axonal regeneration.