Tyrphostin A9 protects axons in experimental autoimmune encephalomyelitis through activation of ERKs.

AIMS: Small molecule compound tyrphostin A9 (A9), an inhibitor of platelet-derived growth factor (PDGF) receptor, was previously reported by our group to stimulate extracellular signal-regulated kinase 1 (ERK1) and 2 (ERK2) in neuronal cells in a PDGF receptor-irrelevant manner. The study aimed to investigate whether A9 could protect axons in experimental autoimmune encephalomyelitis through activation of ERKs. MAIN METHODS: A9 treatment on the protection on neurite outgrowth in SH-SY5Y neuroblastoma cells and primary substantia nigra neuron cultures from the neurotoxin MPP+ were analyzed. Then, clinical symptoms as well as ERK1/2 activation, axonal protection induction, and the abundance increases of the regeneration biomarker GAP-43 in the CNS in the relapsing-remitting experimental autoimmune encephalomyelitis (EAE) model were verified. KEY FINDINGS: A9 treatment could stimulate neurite outgrowth in SH-SY5Y neuroblastoma cells and protect primary substantia nigra neuron cultures from the neurotoxin MPP+. In the relapsing-remitting EAE model, oral administration of A9 successfully ameliorated clinical symptoms, activated ERK1/2, induced axonal protection, and increased the abundance of the regeneration biomarker GAP-43 in the CNS. Interestingly, gene deficiency of ERK1 or ERK2 disrupted the beneficial effects of A9 in MOG-35-55-induced EAE. SIGNIFICANCE: These results demonstrated that small molecule compounds that stimulate persistent ERK activation in vitro and in vivo may be useful in protective or restorative treatment for neurodegenerative diseases.

Centella asiatica Extract Improves Behavioral Deficits in a Mouse Model of Alzheimer's Disease: Investigation of a Possible Mechanism of Action.

Centella asiatica (CA), commonly named gotu kola, is an Ayurvedic herb used to enhance memory and nerve function. To investigate the potential use of CA in Alzheimer's disease (AD), we examined the effects of a water extract of CA (GKW) in the Tg2576 mouse, a murine model of AD with high ß-amyloid burden. Orally administered GKW attenuated ß-amyloid-associated behavioral abnormalities in these mice. In vitro, GKW protected SH-SY5Y cells and MC65 human neuroblastoma cells from toxicity induced by exogenously added and endogenously generated ß-amyloid, respectively. GKW prevented intracellular ß-amyloid aggregate formation in MC65 cells. GKW did not show anticholinesterase activity or protect neurons from oxidative damage and glutamate toxicity, mechanisms of current AD therapies. GKW is rich in phenolic compounds and does not contain asiatic acid, a known CA neuroprotective triterpene. CA thus offers a unique therapeutic mechanism and novel active compounds of potential relevance to the treatment of AD.

Cyclophilin D inactivation protects axons in experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis.

Multiple sclerosis (MS) is the leading cause of neurological disability in young adults, affecting some two million people worldwide. Traditionally, MS has been considered a chronic, inflammatory disorder of the central white matter in which ensuing demyelination results in physical disability [Frohman EM, Racke MK, Raine CS (2006) N Engl J Med 354:942-955]. More recently, MS has become increasingly viewed as a neurodegenerative disorder in which neuronal loss, axonal injury, and atrophy of the CNS lead to permanent neurological and clinical disability. Although axonal pathology and loss in MS has been recognized for >100 years, very little is known about the underlying molecular mechanisms. Progressive axonal loss in MS may stem from a cascade of ionic imbalances initiated by inflammation, leading to mitochondrial dysfunction and energetic deficits that result in mitochondrial and cellular Ca2+ overload. In a murine disease model, experimental autoimmune encephalomyelitis (EAE) mice lacking cyclophilin D (CyPD), a key regulator of the mitochondrial permeability transition pore (PTP), developed EAE, but unlike WT mice, they partially recovered. Examination of the spinal cords of CyPD-knockout mice revealed a striking preservation of axons, despite a similar extent of inflammation. Furthermore, neurons prepared from CyPD-knockout animals were resistant to reactive oxygen and nitrogen species thought to mediate axonal damage in EAE and MS, and brain mitochondria lacking CyPD sequestered substantially higher levels of Ca2+. Our results directly implicate pathological activation of the mitochondrial PTP in the axonal damage occurring during MS and identify CyPD, as well as the PTP, as a potential target for MS neuroprotective therapies.

Antigen-specific therapy promotes repair of myelin and axonal damage in established EAE.

Inflammation results in CNS damage in multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE), an animal model of MS. It is uncertain how much repair of injured myelin and axons can occur following highly selective anti-inflammatory therapy in EAE and MS. In this study, SJL/J mice with established EAE were treated successfully with an antigen-specific recombinant T cell receptor ligand (RTL), RTL401, a mouse I-A(s)/PLP-139-151 construct, after the peak of EAE. To define the mechanisms by which late application of RTL401 inhibits EAE, we evaluated mice at different time points to assess the levels of neuroinflammation and myelin and axon damage in their spinal cords. Our results showed that RTL401 administered after the peak of acute EAE induced a marked reduction in inflammation in the CNS, associated with a significant reduction of demyelination, axonal loss and ongoing damage. Electron microscopy showed that RTL-treated mice had reduced pathology compared with mice treated with vehicle and mice at the peak of disease, as demonstrated by a decrease in continued degeneration, increase in remyelinating axons and the presence of an increased number of small, presumably regenerative axonal sprouts. These findings indicate that RTL therapy targeting encephalitogenic T cells may promote CNS neuroregenerative processes.

Neuroimmunophilin ligands improve functional recovery and increase axonal growth after spinal cord hemisection in rats.

We have previously shown that FK506 accelerates the rate of nerve regeneration in the peripheral nervous system (PNS) and increases regeneration of central nervous system (CNS) axons into a peripheral nerve graft. In the present study, we examined whether FK506 and a nonimmunosuppressive derivative (FK1706) improve functional recovery and long distance regeneration following a hemisection lesion of spinal cord at T10/T11. Rats were given daily subcutaneous injections of either FK506 (2 mg/kg/day), FK1706 (2 mg/kg/day), an equivalent volume of saline or 30% DMSO as vehicle, respectively. Functional recovery was assessed using a modified Tarlov/Klinger scale, walking along progressively narrower wooden beams (7.7-1.7 cm widths), and analysis of footprints obtained during walking. Compared to both control groups, FK506 and FK1706-treated animals demonstrated significant functional recovery 4 days (beam walking), 2 weeks (footprints), and 4 weeks (Tarlov/Klinger scale). By 11 weeks, FK506-treated and FK1706-treated animals were able to walk, albeit poorly, along even the narrowest (1.7 cm) beam. At 11 weeks, the spinal cords were re-exposed and a small piece of gel foam-soaked Fluoro-Gold was placed on the injured side 2-cm caudal to the first injury. Five days later, the animals were perfused and tissues prepared for fluorescence microscopy. FK506-treated and FK1706-treated rats demonstrate a significantly greater number of retrogradely labeled neurons in the red nucleus. The results implicate a nonimmunosuppressant mechanism in FK506's action and suggest that FK506 or a nonimmunosuppressant derivative may be useful for treatment of spinal cord injuries.

Centella asiatica accelerates nerve regeneration upon oral administration and contains multiple active fractions increasing neurite elongation in-vitro.

Axonal regeneration is important for functional recovery following nerve damage. Centella asiatica Urban herb, also known as Hydrocotyle asiatica L., has been used in Ayurvedic medicine for centuries as a nerve tonic. Here, we show that Centella asiatica ethanolic extract (100 microg mL-1) elicits a marked increase in neurite outgrowth in human SH-SY5Y cells in the presence of nerve growth factor (NGF). However, a water extract of Centella was ineffective at 100 microg mL-1. Sub-fractions of Centella ethanolic extract, obtained through silica-gel chromatography, were tested (100 microg mL-1) for neurite elongation in the presence of NGF. Greatest activity was found with a non-polar fraction (GKF4). Relatively polar fractions (GKF10 to GKF13) also showed activity, albeit less than GKF4. Thus, Centella contains more than one active component. Asiatic acid (AA), a triterpenoid compound found in Centella ethanolic extract and GKF4, showed marked activity at 1 microM (microg mL-1). AA was not present in GKF10 to GKF13, further indicating that other active components must be present. Neurite elongation by AA was completely blocked by the extracellular-signal-regulated kinase (ERK) pathway inhibitor PD 098059 (10 microM). Male Sprague-Dawley rats given Centella ethanolic extract in their drinking water (300-330 mg kg-1 daily) demonstrated more rapid functional recovery and increased axonal regeneration (larger calibre axons and greater numbers of myelinated axons) compared with controls, indicating that the axons grew at a faster rate. Taken together, our findings indicate that components in Centella ethanolic extract may be useful for accelerating repair of damaged neurons.

FK 506 reduces tissue damage and prevents functional deficit after spinal cord injury in the rat.

We examined the efficacy of FK 506 in reducing tissue damage after spinal cord injury in comparison to methylprednisolone (MP) treatment. Rats were subjected to a photochemical injury (T8) and were given a bolus of MP (30 mg/kg), FK 506 (2 mg/kg), or saline. An additional group received an initial bolus of FK 506 (2 mg/kg) followed by daily injections (0.2 mg/kg intraperitoneally). Functional recovery was evaluated using open-field walking, inclined plane tests, motor evoked potentials (MEPs), and the H-reflex response during 14 days postoperation (dpo). Tissue sparing and glial fibrillary acidic protein (GFAP), biotinylated tomato lectin LEC, cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and interleukin 1 beta (IL-1 beta) immunoreactivity were quantified in the injured spinal cord. FK 506-treated animals demonstrated significantly better neurologic outcome, higher MEP amplitudes, and lower H-wave amplitude compared to that of saline-treated rats. In contrast, administration of MP did not result in significant differences with respect to the saline-treated group. Histologic examination revealed that tissue sparing was largest in FK 506-treated compared to saline and MP-treated animals. GFAP and COX-2 reactivity was decreased in animals treated with FK 506 compared to that in animals given MP or saline, whereas IL-1 beta expression was similarly reduced in both FK 506- and MP-treated groups. Microglia/macrophage response was reduced in FK 506 and MP-injected animals at 3 dpo, but only in MP-treated animals at 7 dpo with respect to saline-injected rats. Repeated administrations of FK 506 improved functional and histologic results to a greater degree than did a single bolus of FK 506. The results indicate that FK 506 administration protects the damaged spinal cord and should be considered as potential therapy for treating spinal cord injuries.

Coordinate control of axon defasciculation and myelination by laminin-2 and -8.

Schwann cells form basal laminae (BLs) containing laminin-2 (Ln-2; heterotrimer alpha2beta1gamma1) and Ln-8 (alpha4beta1gamma1). Loss of Ln-2 in humans and mice carrying alpha2-chain mutations prevents developing Schwann cells from fully defasciculating axons, resulting in partial amyelination. The principal pathogenic mechanism is thought to derive from structural defects in Schwann cell BLs, which Ln-2 scaffolds. However, we found loss of Ln-8 caused partial amyelination in mice without affecting BL structure or Ln-2 levels. Combined Ln-2/Ln-8 deficiency caused nearly complete amyelination, revealing Ln-2 and -8 together have a dominant role in defasciculation, and that Ln-8 promotes myelination without BLs. Transgenic Ln-10 (alpha5beta1gamma1) expression also promoted myelination without BL formation. Rather than BL structure, we found Ln-2 and -8 were specifically required for the increased perinatal Schwann cell proliferation that attends myelination. Purified Ln-2 and -8 directly enhanced in vitro Schwann cell proliferation in collaboration with autocrine factors, suggesting Lns control the onset of myelination by modulating responses to mitogens in vivo.

Non-FK506-binding protein-12 neuroimmunophilin ligands increase neurite elongation and accelerate nerve regeneration.

Neurotrophic activity of neuroimmunophilin ligands (FK506 and its nonimmunosuppressant derivatives) has been assumed to be mediated by the FK506-binding protein-12 (FKBP-12). We recently showed that activity is retained in hippocampal neurons from FKBP-12 knockout mice, indicating that binding to FKBP-12 is not necessary. Here we show that three nonimmunosuppressant FK506 derivatives (V-13,450, V-13,629, and V-13,670) that do not bind FKBP-12 (>12.5 mM affinity) are equipotent to FKBP-12 ligands (FK506, V-10,367, and V-13,449) for increasing neurite elongation in SH-SY5Y cells. One non-FKBP-12 ligand (V-13,670) is also shown to accelerate functional recovery and nerve regeneration in the rat sciatic nerve crush model. Surprisingly, it exhibited an unusual dose-response effect upon oral administration, showing a novel bimodal dose-response for behavioral functional recovery and myelination, but not for axonal size, suggesting both Schwann cell and neuronal targets. Orally active non-FKBP-12 neuroimmunophilin ligands may be useful for the treatment of human neurological disorders without any potential side effects resulting from FKBP-12 binding.

FK1706, a novel non-immunosuppressive immunophilin: neurotrophic activity and mechanism of action.

Immunophilin ligands are neuroregenerative agents, characterized by binding to FK506 binding proteins (FKBPs), which stimulate recovery of neurons in a variety of injury paradigms. Here we report the discovery of a novel, non-immunosuppressive immunophilin ligand, FK1706. FK1706, a derivative of FK506, showed similarly high affinity for two FKBP subtypes, FKBP-12 and FKBP-52, but inhibited T-cell proliferation and interleukin-2 cytokine production with much lower potency and efficacy than FK506. FK1706 (0.1 to 10 nM) significantly potentiated nerve growth factor (NGF)-induced neurite outgrowth in SH-SY5Y cells, as did FK506. This neurite potentiation could be blocked by an anti-FKBP-52 antibody, as well as by specific pharmacological inhibitors of phospholipase C (PLC), phosphatidylinositol 3-kinase (PI3K), and the Ras/Raf/Mitogen-Activated Protein Kinase (MAPK) signaling pathway. FK1706 also potentiated NGF-induced MAPK activation, with a similar dose-dependency to that necessary for potentiating neurite outgrowth. Taken together, these data suggest that FK1706 is a non-immunosuppressive immunophilin ligand with significant neurotrophic effects, putatively mediated via FKBP-52 and the Ras/Raf/MAPK signaling pathway, and therefore that FK1706 may have therapeutic potential in a variety of neurological disorders.

FK506 and a nonimmunosuppressant derivative reduce axonal and myelin damage in experimental autoimmune encephalomyelitis: neuroimmunophilin ligand-mediated neuroprotection in a model of multiple sclerosis.

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS) in which demyelination and axonal loss result in permanent neurologic disability. We examined the neuroprotective property of the immunosuppressant FK506 (tacrolimus), FK1706 (a nonimmunosuppressant FK506 derivative) and cyclosporin A (CsA) in a chronic relapsing experimental autoimmune encephalomyelitis (EAE) model of MS. Female SJL/J mice were immunized by subcutaneous (s.c.) injection with proteolipid protein 139-151 peptide in complete Freund's adjuvant. At the onset of paralysis, 12-14 days after immunization, mice received daily s.c. injections of FK506 (0.2, 1, and 5 mg/kg), FK1706 (5 mg/kg), CsA (2, 10, and 50 mg/kg), saline or vehicle (30% dimethylsulfoxide) for 30 days. FK506 (at a dose of 5 mg/kg) reduced the severity of the initial disease and suppressed relapses. FK1706 did not significantly alter the clinical course and CsA (at a dose of 50 mg/kg) lessened the severity of the initial episode of EAE but did not alter relapses. In the thoracic spinal cord, FK506 (5 mg/kg), FK1706 (5 mg/kg), and CsA (50 mg/kg) significantly (P < 0.001) reduced the extent of damage in the dorsal, lateral, and ventral white matter by a mean of up to 95, 68, and 30%, respectively. A nonimmunosuppressant dose of FK506 (0.2 mg/kg) also significantly (P < 0.001) reduced the extent of damage in the spinal cord by a mean of up to 45%. Other dosages of these compounds were ineffective. FK506 markedly protects against demyelination and axonal loss in this MS model through immunosuppression and neuroprotection.

FK506 enhances regeneration of axons across long peripheral nerve gaps repaired with collagen guides seeded with allogeneic Schwann cells.

We assessed the effects of FK506 administration on regeneration after a 6-mm gap repair with a collagen guide seeded with allogeneic Schwann cells (SCs) in the mouse sciatic nerve. SCs were isolated from predegenerated adult sciatic nerves and expanded in culture using a defined medium, before being seeded in the collagen guide embedded in Matrigel. Functional reinnervation was evaluated by noninvasive methods to determine recovery of motor, sensory, and autonomic functions in the hindpaw over 4 months postoperation. Histological analysis of the regenerated nerves was performed at the end of the study. Using simple collagen guides for tubulization repair, treatment with an immunosuppressant dose of FK506 (5 mg/kg/day) resulted in significant improvement of the onset and the degree of reinnervation. While the introduction of allogeneic SCs did not improve regeneration versus a collagen guide filled only with Matrigel, treatment with FK506 allowed for successful regeneration in all the mice and for significant improvement in the levels of functional recovery. Compared with the untreated group, there was greater survival of transplanted pre-labeled SCs in the FK506-treated animals. Morphologically, the best nerve regeneration (in terms of nerve caliber and numbers of myelinated axons) was obtained with SC-seeded guides from FK506-treated animals. Thus, FK506 should be considered as adjunct therapy for various types of tubulization repair.

Neuroregenerative and neuroprotective actions of neuroimmunophilin compounds in traumatic and inflammatory neuropathies.

FK506 (tacrolimus, Prograf is an immunosuppressant drug that also has profound neuroregenerative and neuroprotective actions independent of its immunosuppressant activity. The separation of these properties has led to the development of non-immunosuppressant derivatives that retain the neurotrophic activity. This review focuses on the peripheral nerve actions of these compounds following mechanical injury (nerve crush or transection with graft repair) and in models of inflammatory neuropathies. Whereas FK506 may be indicative for the treatment of inflammatory neuropathies where its immunosuppressive action would be advantageous, non-immunosuppressant derivatives represent a new class of potential therapeutic agents for the treatment of human neurological conditions in general. Moreover, these studies have led to the discovery of a novel mechanism whereby these compounds activate intrinsic neuroregenerative and neuroprotective pathways in the neuron.

Comparison of continuous and discontinuous FK506 administration on autograft or allograft repair of sciatic nerve resection.

An immunosuppressant drug that also possesses neuroregenerative properties, FK506 enhances the rate of axonal regeneration and improves recovery after nerve lesions. Nevertheless, prolonged immunosuppression may not be justified to assure the success of nerve regeneration. In this study, we compare the effects of continuous and discontinuous FK506 treatment on regeneration and reinnervation after sciatic nerve resection repaired with autologous or allogenic grafts in the mouse. For each type of repair, one group received FK506 (5 mg/kg) for 4 months, whereas a second group was treated with FK506 at 5 mg/kg for 5 weeks followed by 3 mg/kg for 4 weeks; a control group received saline only. Functional reinnervation was assessed by noninvasive methods to determine recovery of motor, sensory, and autonomic functions in the hind paw over 4 months after operation. Morphological analysis of the regenerated nerves was performed at the termination of the study. Autografts and allografts treated with sustained FK506 (5 mg/kg) reached high levels of reinnervation and followed a course of recovery faster than controls. The numbers of myelinated fibers also were similar. Allografts without immunosuppression demonstrated a slower rate of regeneration, exhibiting lower final levels of recovery compared with other groups and containing fewer numbers of regenerating myelinated fibers. Withdrawal of immunosuppressant therapy resulted in a decline in the degree of reinnervation in all functions tested during the third month, with stabilization between the third and fourth months. The number of regenerated myelinated fibers in the group was significantly lower than in autografts. Thus, continuous or discontinuous FK506 administration slightly accelerated the rate of reinnervation in autografts. In allograft repair, FK506 significantly enhanced both the rate and degree of regeneration and recovery, but its withdrawal resulted in graft rejection, a marked deterioration in function, and loss of regenerating fibers.

The immunosuppressant FK506 elicits a neuronal heat shock response and protects against acrylamide neuropathy.

Acrylamide (AC) is a known industrial neurotoxic chemical that has been recently found in carbohydrate-rich foods cooked at high temperatures. Repeated AC administration produces a pronounced neuropathy characterized by flaccid paralysis and ataxia and represents a well-established animal model of progressive axonal loss. AC also elicits prominent morphologic alterations (e.g., eccentrically placed nuclei, infolding of the nuclear membrane, accumulations of dense bodies, and clusters of smooth endoplasmic reticulum (SER) associated with numerous microtubules) in cerebellar Purkinje cells that may contribute to the pronounced ataxia in these animals. Here, we examined the neuroprotective action of FK506 (tacrolimus) in male and female rats given daily intraperitoneal injections of AC (30 mg/kg) for 4 weeks. Daily subcutaneous injections of FK506 (2 mg/kg/day) dramatically reduced the behavioral signs of neuropathy (i.e., paralysis and ataxia), markedly protected against axonal loss (by 82% and 73% in the tibial nerves of male and female rats, respectively), and reduced the pathologic changes in Purkinje cells. In a separate study, subcutaneous injections of FK506 (2 or 10 mg/kg) for 2 weeks markedly increased heat shock protein-70 (Hsp-70) immunostaining in sensory neurons, motor neurons, Purkinje cells, and other regions of the brain (in particular, the amygdala) from nonintoxicated and AC-intoxicated rats compared to controls. In contrast, AC-intoxicated animals not given FK506 demonstrated reduced Hsp-70 staining. Thus, the ability of FK506 to increase Hsp-70 expression may underlie its neuroprotective action. We suggest that compounds capable of eliciting a heat shock response may be useful for the treatment of human neuropathies.

FK506 requires stimulation of the extracellular signal-regulated kinase 1/2 and the steroid receptor chaperone protein p23 for neurite elongation.

The immunosuppressant drug FK506 (tacrolimus) accelerates nerve regeneration in vivo and increases neurite elongation in vitro. We have proposed that the mechanism involves binding to the FK506-binding protein 52, a chaperone component of mature steroid receptor complexes, and a subsequent 'gain-of-function' involving p23 dissociation from Hsp-90 in the complex and extracellular signal-regulated kinase (ERK) activation. Here, we tested the involvement of the ERK and p23 in neurite elongation by FK506 in human SH-SY5Y cells. FK506 (10 nM) increased ERK1/2 phosphorylation at 12 and 24 h, eliciting a 3.5-fold increase at 24 h, which was inhibited in a concentration-dependent manner by an antibody (JJ3) to recombinant human p23. Neurite elongation by FK506 (10 nM), determined by measuring neurite lengths at 96 and 168 h, was completely blocked by the mitogen-activated protein kinase inhibitor PD 098059 (10 microM) and prevented, in a concentration-dependent fashion, by the p23 antibody. Taken together, the results demonstrate the functional role for ERK and p23 in the neurite elongation activity of FK506 and reveal a novel signal transduction pathway involving p23 activation of ERK. We suggest that compounds that stimulate or mimic p23 may be useful for accelerating nerve regeneration.

FK506 enhances reinnervation by regeneration and by collateral sprouting of peripheral nerve fibers.

We examined the effects of FK506 administration on the degree of target reinnervation by regenerating axons (following sciatic nerve crush) and by collateral sprouts of the intact saphenous nerve (after sciatic nerve resection) in the mouse. FK506-treated animals received either 0.2 or 5 mg/kg/day, dosages previously found to maximally increase the rate of axonal regeneration in the mouse. Functional reinnervation of motor, sensory, and sweating activities was assessed by noninvasive methods in the hind paw over a 1-month period following lesion. Morphometric analysis of the regenerated nerves and immunohistochemical labeling of the paw pads were performed at the end of follow-up. In the sciatic nerve crush model, FK506 administration shortened the time until target reinnervation and increased the degree of functional and morphological reinnervation achieved. The recovery achieved by regeneration was greater overall with the 5 mg/kg dose than with the dose of 0.2 mg/kg of FK506. In the collateral sprouting model, reinnervation by nociceptive and sudomotor axons was enhanced by FK506. Here, the field expansion followed a faster course between 4 and 14 days in FK506-treated animals. In regard to dose, while collateral sprouting of nociceptive axons was similarly increased at both dosages (0.2 and 5 mg/kg), sprouting of sympathetic axons was more extensive at the high dose. This suggests that the efficacy of FK506 varies between subtypes of neurons. Taken together, our findings indicate that, in addition to an effect on rate of axonal elongation, FK506 improves functional recovery of denervated targets by increasing both regenerative and collateral reinnervation.

Comparative dose-dependence study of FK506 on transected mouse sciatic nerve repaired by allograft or xenograft.

We evaluated the effects of FK506, at doses of 0.2, 2, and 5 mg/kg/day, on the response to nerve grafts implanted in outbred mice. A 6 mm long segment of the sciatic nerve was transected and repaired by autograft (the same segment resected), allograft (from another mouse), or xenograft (from a rat nerve). The regenerating nerves were harvested after 3 weeks and studied under light and electron microscope. Allografts of animals treated with the 5 mg/kg/day dose of FK506 appeared similar to those from autografts, demonstrating an equivalent number of myelinated fibers. In mice treated with the 2 mg/kg/day dose, regeneration was slightly hindered, as indicated by the reduced number of myelinated fibers. In contrast, in mice given a 0.2 mg/kg/day dose of FK506, allografts were not different from untreated allografts; both groups showed a marked rejection response with only few unmyelinated axons and no myelinated fibers. Xenografts showed a more severe rejection than allografts, with a marked inflammatory cell reaction throughout the graft. In contrast, in mice treated with the 5 mg/kg/day dose, xenografts exhibited a mild cell reaction and a greater number of regenerated myelinated fibers. In conclusion, effective axonal regeneration is achieved with FK506 administration at doses of 5 mg/kg/day through allografts and, partially, through xenografts.

Neuroimmunophilin ligands: the development of novel neuroregenerative/ neuroprotective compounds.

FK506 (tacrolimus), initially developed as an immunosuppressant drug, represents a class of compounds with potential high impact for the treatment of human neurological disorders. While immunosuppression is mediated by the 12-kD FK506-binding-protein (FKBP-12), the neurite elongation activity of FK506 involves FKBP-52 (also known as FKBP-59 or Hsp-56), a component of mature steroid receptor complexes: FKBP-52 binds to Hsp-90, which bind to p23 and the steroid receptor protein to form the complex. The brief review focuses on how three classes of compounds (FK506 derivatives, steroid hormones, and ansamycin anti-cancer drugs, e.g., geldanamycin) increase neurite elongation/nerve regeneration (axonal elongation). A model is presented whereby neurite elongation is elicited by compounds that bind to steroid receptor chaperone proteins (e.g., FKBP-52 and Hsp-90) and thereby disrupt mature steroid receptor complexes (comprising FKBP-52, Hsp-90 and p23 in addition to the steroid receptor binding protein). Disruption of the complex leads to a "gain-of-function" whereby one or more of these steroid receptor chaperone proteins (i.e, FKBP-52, Hsp-90 or p23) activates mitogen-associated protein (MAP) kinase/extracellular signal-regulated kinase (ERK) pathway. Thus, the neurotrophic actions of these distinct classes of compounds can be understood from their ability to bind steroid receptor chaperones, thereby providing a unique receptor-mediated means to activate the ERK pathway. These studies thereby shed new light on the intrinsic mechanism regulating axonal elongation. Furthermore, this mechanism may also underlie calcineurin-independent neuroprotective actions of FK506. We suggest that components of steroid receptor complexes are novel targets for the design of neuroregenerative/neuroprotective drugs.