tacrolimus and Nerve-Degeneration

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.

Topics: Animals; Axons; Graft Survival; Humans; Immunophilins; Inflammation; Models, Neurological; Nerve Crush; Nerve Degeneration; Nerve Regeneration; Neuroprotective Agents; Peripheral Nervous System Diseases; Tacrolimus; Wound Healing

Tacrolimus, a calcineurin (CaN) inhibitor, has been used for treatment of refractory allergic ocular disease, although its role in optic nerve degeneration remains to be elucidated. In this study, we investigated whether tacrolimus modulates tumor necrosis factor (TNF)-mediated axonal degeneration and whether it alters nuclear factor of activated T cells (NFATc), a downstream effector of CaN signaling. Immunoblot analysis showed no significant difference in CaNAα protein levels in optic nerve on day 3, 7, or 14 after TNF injection compared with PBS injection. However, a significant increase in NFATc1 protein level was observed in optic nerve 7 days after TNF injection. This increase was negated by simultaneous administration of tacrolimus. Administration of tacrolimus alone did not change the NFATc1 protein level in comparison to that observed after PBS injection. A significant increase in TNF protein level was observed in optic nerve 14 days after TNF injection and this increase was prevented by tacrolimus. Immunohistochemical analysis showed the immunoreactivity of NFATc1 to be increased in optic nerve after TNF injection. This increased immunoreactivity was colocalized with glial fibrillary acidic protein and was suppressed by tacrolimus. Treatment of tacrolimus significantly ameliorated the TNF-mediated axonal loss. These results suggest that tacrolimus is neuroprotective against axon loss in TNF-induced optic neuropathy and that the effect arises from suppression of the CaN/NFATc1 pathway.

Topics: Animals; Axons; Calcineurin Inhibitors; Male; Nerve Degeneration; Neuroprotective Agents; Optic Nerve; Optic Nerve Diseases; Rats, Wistar; Signal Transduction; Tacrolimus; Transcription Factors; Tumor Necrosis Factor-alpha

Herpes simplex virus type-1 (HSV-1) is a leading cause of neurotrophic keratitis, characterized by decreased or absent corneal sensation due to damage to the sensory corneal innervation. We previously reported the elicited immune response to infection contributes to the mechanism of corneal nerve regression/damage during acute HSV-1 infection. Our aim is to further establish the involvement of infiltrated macrophages in the mechanism of nerve loss upon infection.. Macrophage Fas-Induced Apoptosis (MAFIA) transgenic C57BL/6 mice were systemically treated with AP20187 dimerizer or vehicle (VEH), and their corneas, lymph nodes, and blood were assessed for CD45+CD11b+GFP+ cell depletion by flow cytometry (FC). Mice were ocularly infected with HSV-1 or left uninfected. At 2, 4, and/or 6 days post infection (PI), corneas were assessed for sensitivity and harvested for FC, nerve structure by immunohistochemistry, viral content by plaque assay, soluble factor content by suspension array, and activation of signaling pathways by Western blot analysis. C57BL6 mice were used to compare to the MAFIA mouse model.. MAFIA mice treated with AP20187 had efficient depletion of CD45+CD11b+GFP+ cells in the tissues analyzed. The reduction of CD45+CD11b+GFP+ cells recruited to the infected corneas of AP20187-treated mice correlated with preservation of corneal nerve structure and function, decreased protein concentration of inflammatory cytokines, and decreased STAT3 activation despite no changes in viral content in the cornea compared to VEH-treated animals.. Our results suggest infiltrated macrophages are early effectors in the nerve regression following HSV-1 infection. We propose the neurodegeneration mechanism involves macrophages, local up-regulation of IL-6, and activation of STAT3.

Topics: Animals; Blotting, Western; Cornea; Disease Models, Animal; Flow Cytometry; Herpesvirus 1, Human; Immunohistochemistry; Interleukin-6; Keratitis, Herpetic; Macrophages; Mice; Mice, Inbred C57BL; Mice, Transgenic; Nerve Degeneration; Receptor, Macrophage Colony-Stimulating Factor; STAT3 Transcription Factor; Tacrolimus; Trigeminal Nerve; Trigeminal Nerve Diseases; Viral Plaque Assay

HIV-associated neurocognitive disorders (HAND) continue to be a common morbidity associated with chronic HIV infection. It has been shown that HIV proteins (e.g., gp120) released from infected microglial/macrophage cells can cause neuronal damage by triggering inflammation and oxidative stress, activating aberrant kinase pathways, and by disrupting mitochondrial function and biogenesis. Previous studies have shown that FK506, an immunophilin ligand that modulates inflammation and mitochondrial function and inhibits calcineurin, is capable of rescuing the neurodegenerative pathology in models of Parkinson's disease, Alzheimer's disease, and Huntington's disease. In this context, the main objective of this study was to evaluate if FK506 could rescue the neuronal degeneration and mitochondrial alterations in a transgenic (tg) animal model of HIV1-gp120 neurotoxicity.. GFAP-gp120 tg mice were treated with FK506 and analyzed for neuropathology, behavior, mitochondrial markers, and calcium flux by two-photon microscopy.. We found that FK506 reduced the neuronal cell loss and neuro-inflammation in the gp120 tg mice. Moreover, while vehicle-treated gp120 tg mice displayed damaged mitochondria and increased neuro-inflammatory markers, FK506 rescued the morphological mitochondrial alterations and neuro-inflammation while increasing levels of optic atrophy 1 and mitofusin 1. By two-photon microscopy, calcium levels were not affected in the gp120 tg mice and no effects of FK506 were detected. However, at a functional level, FK506 ameliorated the gp120 tg mice hyperactivity in the open field.. Together, these results suggest that FK506 might be potentially neuroprotective in patients with HAND by mitigating inflammation and mitochondrial alterations.

Topics: Analysis of Variance; Animals; Calcium; Calcium-Binding Proteins; Disease Models, Animal; Encephalitis; HIV Envelope Protein gp120; Immunosuppressive Agents; Interleukin-6; Mice; Microfilament Proteins; Mitochondria; Nerve Degeneration; Nerve Tissue Proteins; Neurotoxicity Syndromes; Tacrolimus; Tacrolimus Binding Proteins; Treatment Outcome

Cerebral ischemia in the territory of the middle cerebral artery (MCA) can induce delayed neuronal cell death in the ipsilateral substantia nigra (SN) remote from the primary ischemic lesion. This exofocal postischemic neuronal degeneration (EPND) may worsen stroke outcomes. However, the mechanisms leading to EPND are poorly understood. Here, we studied the time course of EPND via sequential magnetic resonance imaging (MRI) and immunohistochemistry for up to 28 days after 30 minutes' occlusion of the MCA (MCAo) and reperfusion in the mouse. Furthermore, the effects of delayed treatment with FK506 and MK-801 on the development of EPND were investigated. Secondary neuronal degeneration in the SN occurred within the first week after MCAo and was characterized by a marked neuronal cell loss on histology. Sequential neuroimaging examinations revealed transient MRI changes, which were detectable as early as day 4 after MCAo and thus heralding histologic evidence of EPND. Treatment with MK-801, an established anti-excitotoxic agent, conferred protection against EPND even when initiated days after the initial ischemic event, which was not evident with FK506. Our findings define a secondary time window for delayed neuroprotection after stroke, which may provide a promising target for the development of novel therapies.

Topics: Animals; Brain Ischemia; Cell Count; Dizocilpine Maleate; Immunohistochemistry; Immunosuppressive Agents; Infarction, Middle Cerebral Artery; Magnetic Resonance Imaging; Male; Mice; Mice, 129 Strain; Nerve Degeneration; Neuroprotective Agents; Substantia Nigra; Tacrolimus

Traumatic brain injury (TBI) causes both an acute loss of tissue and a progressive injury through reactive processes such as excitotoxicity and inflammation. These processes may worsen neural dysfunction by altering neuronal circuitry beyond the focally-damaged tissue. One means of circuit alteration may involve dendritic spines, micron-sized protuberances of dendritic membrane that support most of the excitatory synapses in the brain. This study used a modified Golgi-Cox technique to track changes in spine density on the proximal dendrites of principal cells in rat forebrain regions. Spine density was assessed at 1 h, 24 h, and 1 week after a lateral fluid percussion TBI of moderate severity. At 1 h after TBI, no changes in spine density were observed in any of the brain regions examined. By 24 h after TBI, however, spine density had decreased in ipsilateral neocortex in layer II and III and dorsal dentate gyrus (dDG). This apparent loss of spines was prevented by a single, post-injury administration of the calcineurin inhibitor FK506. These results, together with those of a companion study, indicate an FK506-sensitive mechanism of dendritic spine loss in the TBI model. Furthermore, by 1 week after TBI, spine density had increased substantially above control levels, bilaterally in CA1 and CA3 and ipsilaterally in dDG. The apparent overgrowth of spines in CA1 is of particular interest, as it may explain previous reports of abnormal and potentially epileptogenic activity in this brain region.

Topics: Animals; Brain; Brain Injuries; Dendritic Spines; Disease Models, Animal; Male; Nerve Degeneration; Rats; Rats, Sprague-Dawley; Tacrolimus

Neurodegenerative diseases and neurotraumas typically result in apoptosis of specific neurons leading to the pathology observed during the disease state. Existing treatments target the symptoms instead of preventing the death of these neurons. Although neuroprotective drugs should be useful as a treatment to prevent further loss of neurons, efficacious molecules are lacking. FK506 (tacrolimus), a widely used immunosuppressant drug, has significant neuroprotective and neuroregenerative properties throughout the central nervous system, including the eye. FK506 achieves these properties through interaction with FK506 binding proteins (FKBP), including FK506 binding protein 51 (FKBP51). In this study, we examine the effects of FKBP51 as a neuroprotective agent on a neuronal cell line.. We cultured 661w cell cultures with or without FK506, or stably transfected them with an FKBP51 expression vector. These cells were then exposed to the apoptosis-inducing agent staurosporine. Cell viability was determined using a calcein AM/propidium iodide assay. Protein levels and activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) were determined by western immunoblot analysis.. FKBP51 overexpression significantly protected 661w cell cultures from staurosporine-induced apoptosis. FKBP51 overexpression also significantly increased NF-κB p65 protein levels and activated NF-κB p65. FK506 treatment significantly protected 661w neuronal cultures from staurosporine-induced apoptosis. FK506 increased FKBP51, NF-κB p65, and levels of activated NF-κB p65 protein.. These results suggest that FKBP51 protects 661w cell cultures from apoptosis induced by staurosporine. Additionally, FK506 protected 661w cell cultures from apoptosis and displayed a mechanism similar to that of FKBP51 overexpression. Both FK506 and FKBP51 appear to act through activation of NF-κB p65 protein, suggesting a common pathway for neuroprotection. These findings implicate FKBP51 as a protein important to neuronal cell culture survival. FKBP51 may be a potential therapeutic drug target for preventing the neurodegeneration and neurotrauma that occur during neurodegenerative diseases.

Topics: Animals; Apoptosis; Cell Line, Tumor; Cell Survival; Gene Expression; Mice; Nerve Degeneration; Neurons; Neuroprotective Agents; NF-kappa B; Plasmids; Retina; Staurosporine; Tacrolimus; Tacrolimus Binding Proteins; Transfection

Prion diseases are fatal neurodegenerative disorders characterized by a long pre-symptomatic phase followed by rapid and progressive clinical phase. Although rare in humans, the unconventional infectious nature of the disease raises the potential for an epidemic. Unfortunately, no treatment is currently available. The hallmark event in prion diseases is the accumulation of a misfolded and infectious form of the prion protein (PrP(Sc)). Previous reports have shown that PrP(Sc) induces endoplasmic reticulum stress and changes in calcium homeostasis in the brain of affected individuals. In this study we show that the calcium-dependent phosphatase Calcineurin (CaN) is hyperactivated both in vitro and in vivo as a result of PrP(Sc) formation. CaN activation mediates prion-induced neurodegeneration, suggesting that inhibition of this phosphatase could be a target for therapy. To test this hypothesis, prion infected wild type mice were treated intra-peritoneally with the CaN inhibitor FK506 at the clinical phase of the disease. Treated animals exhibited reduced severity of the clinical abnormalities and increased survival time compared to vehicle treated controls. Treatment also led to a significant increase in the brain levels of the CaN downstream targets pCREB and pBAD, which paralleled the decrease of CaN activity. Importantly, we observed a lower degree of neurodegeneration in animals treated with the drug as revealed by a higher number of neurons and a lower quantity of degenerating nerve cells. These changes were not dependent on PrP(Sc) formation, since the protein accumulated in the brain to the same levels as in the untreated mice. Our findings contribute to an understanding of the mechanism of neurodegeneration in prion diseases and more importantly may provide a novel strategy for therapy that is beneficial at the clinical phase of the disease.

Topics: Animals; Behavior, Animal; Brain; Calcineurin Inhibitors; Calcium Signaling; Disease Models, Animal; Disease Progression; Injections, Intraperitoneal; Mice; Mice, Inbred C57BL; Nerve Degeneration; Prion Diseases; Survival Analysis; Tacrolimus

The expression of the neutrophil chemokine macrophage inflammatory protein-2 (MIP-2/CXCL2) and the monocyte chemokine monocyte chemotactic protein-1 (MCP-1/CCL2) have been described in glial cells in vitro but their origin following TBI has not been established. Furthermore, little is known of the modulation of these chemokines. Chemokine expression was investigated in male Sprague-Dawley rats following moderate lateral fluid percussion injury (LFPI). At 0, 4, 8, 12, and 24 h after injury, brains were harvested and MIP-2/CXCL2 and MCP-1/CCL2 levels measured by ELISA. To investigate the inhibition of chemokine expression a second cohort of animals received dexamethasone (1-15mg/kg), FK506 (1mg/kg), or vehicle, systemically, immediately after injury. These animals were sacrificed at the time of peak chemokine expression. A third cohort of animals was also sacrificed at the time of peak chemokine expression and immunohistochemistry performed for MIP-2/CXCL2 and MCP-1/CCL2. Following LFPI, chemokines were increased in the ipsilateral hemisphere, MIP-2/CXCL2 peaking at 4 h and MCP-1/CCL2 peaking at 8-12 h post-injury. Dexamethasone significantly reduced cortical MCP-1/CCL2, but not MIP-2/CXCL2 concentrations. FK506 did not inhibit chemokine expression. In undamaged brain, chemokine expression was localized to cells with a neuronal morphology. For MIP-2/CXCL2 this was supported by double staining for the neuronal antigen NeuN. In contused tissue, increased MIP-2/CXCL2 and MCP-1/CCL2 staining was visible in cells with the morphology of degenerating neurons. MIP-2/CXCL2 and MCP-1/CCL2 are increased after injury, and neurons appear to be the source of this expression. Chemokine expression was selectively inhibited by dexamethasone. The implications of this are discussed.

Topics: Animals; Anti-Inflammatory Agents; Brain; Brain Injuries; Chemokine CCL2; Chemokine CXCL2; Dexamethasone; Disease Models, Animal; DNA-Binding Proteins; Down-Regulation; Immunosuppressive Agents; Male; Nerve Degeneration; Nerve Tissue Proteins; Neurons; Nuclear Proteins; Rats; Rats, Sprague-Dawley; Tacrolimus; Time Factors; Up-Regulation

The immunosuppressant FK506 (1 mg/kg, i.p.) reduces the infarct size following 90 min occlusion of the middle cerebral artery (MCAo) in adult rat brain. Here we have investigated the effect of FK506 on cerebral immune cells that are considered to contribute to neurodegeneration. FK506 substantially attenuated the response of resident and peripheral immune cells following transient ischemia. Between 24 hr and 5 days after MCAo, FK506 reduced the T-cell infiltration in the infarct area as well as the presence of activated and/or phagocytic OX-18, OX-42, GSA-IB4, Iba1, and ED1 positive microglia/macrophages. FK506 also lowered the protein levels of TNFalpha and IL-2 in ischemic brain areas. Repetitive application of FK506 over 20 days attenuated the activation of microglia in the substantia nigra (SN), an area of secondary degeneration. Importantly, FK506 conferred also lasting protection of the neurons of SN; these neurons degenerate by withdrawal of neurotrophic factors from the striatum that undergoes necrotic death as part of the ischemic core. To understand the molecular basis of FK506 effects in cerebral immune cells, we determined in primary postnatal day 0/1 (P0/P1) microglia (i) the expression of the FK506 binding proteins FKBP12, FKBP52, and FKPB65 and (ii) that FK506 (1-100 ng/mL) lowered the number of resting or lipopolysaccharide stimulated microglia as well as we induced the lipopolysaccharide release of TNFalpha in a dose-dependent manner. In summary, FK506 confers rescue of brain tissue following cerebral ischemia not only by neuronal protection, but also by suppression of microglial activation and peripheral immune responses.

Topics: Animals; Biomarkers; Brain Ischemia; Cells, Cultured; Cerebral Infarction; Chemotaxis, Leukocyte; Coculture Techniques; Corpus Striatum; Cytokines; Disease Models, Animal; Encephalitis; Gliosis; Immunosuppressive Agents; Macrophages; Male; Microglia; Nerve Degeneration; Nerve Tissue Proteins; Neuroprotective Agents; Protein Binding; Rats; Rats, Sprague-Dawley; Substantia Nigra; T-Lymphocytes; Tacrolimus

Several kinds of injury models, such as crush, transection and graft repair have been well studied in terms of neuroprotective effect of FK506. However, definitive experimental studies are lacking on focal degeneration or ischemia. In the present study, our goal was to investigate the effect of FK506 on functional recovery of the sciatic nerve after focal ischemia, produced by stripping of the epineurial vessels. A total number of 48 Wistar rats were used for this purpose and divided into four groups (control, sham-operated, FK506-treated, and Vehicle-treated). Sciatic nerves were approached by femoral and gluteal muscle splitting. Then, epineurial vessels around the sciatic nerve were stripped in the FK506-treated and Vehicle-treated groups. After operation, 5mg/kg/day FK506 administration was initiated by subcutaneous injection until animal sacrifice. The same volume of saline was administrated to the vehicle-treated group. The functional and sensory recoveries were tested by walking pattern analysis and pinch test in every postoperative week. The animals were sacrificed in the end of the fourth postoperative week and sciatic nerve samples were harvested and processed for electron microscopic evaluation. Our data revealed that FK506 administration showed beneficial effect on subperineurial degeneration/demyelinization from functional, sensorial, and ultrastructural points of view. The sciatic nerve samples in the FK506-treated group had several remyelinated fibers compared to the vehicle-treated group. Our literature searches revealed that FK506 administration has not, to our knowledge, been studied in focal ischemic degeneration produced by stripping of the epineurial vessels.

Topics: Animals; Demyelinating Diseases; Female; Gait Disorders, Neurologic; Immunosuppressive Agents; Microscopy, Electron; Nerve Degeneration; Neurons; Pain Measurement; Rats; Rats, Wistar; Recovery of Function; Sciatic Nerve; Sensation; Tacrolimus; Walking

Aggregation of alpha-synuclein (alpha-SYN) plays a key role in Parkinson's disease. We have previously shown that aggregation of alpha-SYN in vitro is accelerated by addition of FK506 binding proteins (FKBP) and that this effect can be counteracted by FK506, a specific inhibitor of these enzymes. In this paper, we investigated in detail the effect of FKBP12 on early aggregation and on fibril formation of wild-type, A53T and A30P alpha-SYN. FKBP12 has a much smaller effect on the fibril formation of these two clinical mutants alpha-SYN. Using an inactive enzyme, we were able to discriminate between catalytic and non-catalytic effects that differentially influence the two processes. A model explaining non-linear concentration dependencies is proposed.

Topics: alpha-Synuclein; Amino Acid Substitution; Brain; Catalytic Domain; Cell Line, Tumor; Humans; Immunosuppressive Agents; Mutation; Nerve Degeneration; Neurofibrillary Tangles; Neurons; Nonlinear Dynamics; Parkinson Disease; Tacrolimus; Tacrolimus Binding Protein 1A; Time Factors

Filamentous tau inclusions are hallmarks of Alzheimer's disease (AD) and related tauopathies, but earlier pathologies may herald disease onset. To investigate this, we studied wild-type and P301S mutant human tau transgenic (Tg) mice. Filamentous tau lesions developed in P301S Tg mice at 6 months of age, and progressively accumulated in association with striking neuron loss as well as hippocampal and entorhinal cortical atrophy by 9-12 months of age. Remarkably, hippocampal synapse loss and impaired synaptic function were detected in 3 month old P301S Tg mice before fibrillary tau tangles emerged. Prominent microglial activation also preceded tangle formation. Importantly, immunosuppression of young P301S Tg mice with FK506 attenuated tau pathology and increased lifespan, thereby linking neuroinflammation to early progression of tauopathies. Thus, hippocampal synaptic pathology and microgliosis may be the earliest manifestations of neurodegenerative tauopathies, and abrogation of tau-induced microglial activation could retard progression of these disorders.

Topics: Aging; Amygdala; Amyloid Neuropathies; Animals; Atrophy; Blotting, Western; Brain; Gliosis; Humans; Immunohistochemistry; Immunosuppressive Agents; Macrophage Activation; Mice; Microglia; Microscopy, Electron; Microtubules; Mossy Fibers, Hippocampal; Nerve Degeneration; Neurofibrillary Tangles; Phosphorylation; Solubility; Spinal Cord; Synapses; Tacrolimus; tau Proteins

An in vivo study in Wistar albino rats with injured spinal cord.. Department of Neurosurgery, Biochemistry and Pathology, Gazi University, Ankara, Turkey.. The aim of this study was to investigate and compare the effects of FK506 an immunosupressive agent with methylprednisolone (MP) on lipid peroxidation (LP) in injured spinal cord tissue.. A total of 28 adult healthy Wistar albino rats were subjected to traumatic spinal cord injuries (SCI) by using an aneurysmal clip compression technique, and they were divided into four groups. The G1 group (n=8) received FK506 (1 mg/kg); the G2 group (n=8) received FK506 (1 mg/kg) and MP (30 mg/kg); the G3 group (n=6) received only MP (30 mg/kg); and the G4 group (n=6) received no medication. The injured spinal cord tissue was studied by means of lipid peroxides, malondialdehyde (MDA), with thiobarbituric acid reaction and additionally the FK506 (G1); the MP (G3) groups were studied for histopathologic alterations 72 h after SCI with eight separate animals.. Although LP values of G1, G2, G3 showed no statistical difference between intergroup analyses (P=0.547), a histopathological examination revealed that in the group that received MP, the oedema pattern was more significant than the group that received FK506. Another interesting finding was the presence of polymorphonuclear leucocytes in the MP group, whereas no infiltration was found in the FK506 group.. Analysis of the results indicated that FK506 is a valuable pharmacological agent that could be used to decrease the LP and polymorphonuclear leucocyte infiltration and inflamatory reactions in the injured spinal cord tissue.

Topics: Animals; Chemotaxis, Leukocyte; Disease Models, Animal; Down-Regulation; Drug Synergism; Drug Therapy, Combination; Edema; Free Radicals; Immunosuppressive Agents; Lipid Peroxidation; Male; Malondialdehyde; Methylprednisolone; Nerve Degeneration; Oxidative Stress; Rats; Rats, Wistar; Spinal Cord; Spinal Cord Injuries; Tacrolimus

The mechanism of action of the neurotoxin 6-hydroxydopamine (6-OHDA) is thought to involve the generation of free radicals and subsequent apoptotic processes. We have demonstrated in vitro that the neuroimmunophilin, FK506 (10-100 nM), dose dependently and significantly restored the ROS production to the control level, increased the Bcl-2 protein level, partly inhibited the cytochrome C release from mitochondria and reduced the caspase-3 activation in SH-SY5Y cells. On the other hand, there was no significant restoration of the ATP level by FK506 and the toxin activated proteins, p53 and Bax, were not normalized by FK506. In support of these latter results, daily administration of FK506 for 7 days to rats (0.5, 1 and 3 mg/kg i.p.) did not significantly prevent the apomorphine-induced contralateral circling, measured 2 weeks after unilateral nigral lesioning. Moreover, FK506 pretreatment did not significantly lower the toxin elevated lipid peroxidation levels, indicating that oxidative stress was present even after the FK506 treatment in the lesioned striatum. Taken together, our results with FK506 are inconsistent. We confirm the antioxidant nature of FK506, that is, it blocks ROS production in SH-SY5Y cells. However, there were no significant protective effects in any apoptotic analyses in SH-SY5Y cells and in animal studies, a 7-day FK506 pre-treatment was not able to reverse the toxic effect of 6-OHDA in a rat model of Parkinson's disease.

Topics: Animals; Apomorphine; Behavior, Animal; Caspase 3; Caspases; Cell Death; Cell Line, Tumor; Disease Models, Animal; Dopamine; Dopamine Agonists; Dose-Response Relationship, Drug; Immunophilins; Male; Motor Activity; Nerve Degeneration; Neuroblastoma; Neurons; Neuroprotective Agents; Oxidopamine; Parkinson Disease; Random Allocation; Rats; Rats, Wistar; Tacrolimus

Large scale screening for neuroprotective drugs for peripheral neuropathies requires development of a high throughput system that is reliable and reproducible. Currently most accurate outcome measures of axonal degeneration are based on time-consuming, laborious measurement of morphological changes in neurites. In order to improve on the scalability of the screening procedure we developed a real-time RT-PCR based method of gene expression that correlates very well with morphological measures of neuritic degeneration. We examined the changes in GAP-43 expression in primary dorsal root ganglion (DRG) neurons in vitro with exposure to a zalcitabine (ddC), an antiretroviral drug that causes neuropathy in human immunodeficiency virus (HIV)-infected individuals, with and without FK506, an immunophilin ligand with neuroprotective and neuroregenerative properties. Similar to morphological measures of neuritic degeneration, in ddC-treated cultures there was a reduction in the expression of GAP-43 mRNA. This was prevented, in a dose-dependent manner, by co-administration of FK506. This assay, performed in a 96-well format, can easily be scaled for high throughput screening (HTS) using robotic systems.

Topics: Animals; Animals, Newborn; Cell Culture Techniques; Cells, Cultured; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Fetus; Ganglia, Spinal; GAP-43 Protein; Gene Expression; Nerve Degeneration; Neurons; Neuroprotective Agents; Rats; Reproducibility of Results; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tacrolimus; Zalcitabine

The mitochondrial toxin 3-nitropropionic acid (3-NP) has been largely used to study neurodegenerative disorders in which bioenergetic defects are implicated. In the present study, we analyzed the molecular pathways involved in FK506 neuroprotection against cell death induced by 3-NP, using cultured cortical neurons. 3-NP induced cytochrome c release and increased caspases -2, -3, -8, and -9-like activities, although, calpain activity was not significantly affected. FK506 decreased cytochrome c release and caspase-3-like activity induced by 3-NP, without changing the activities of other caspases. FK-506 also decreased the number of apoptotic neurons, determined by Hoechst. Under these conditions, FK506 alone significantly reduced calcineurin activity by about 50%. Our results also showed a decrease in mitochondrial Bax and an increase in mitochondrial Bcl-2 levels upon exposure to FK506 and 3-NP. However, no significant changes occurred in total Bcl-2 and Bax levels. Altogether, the results suggest that FK506 neuroprotection against 3-NP-induced apoptosis is associated with the redistribution of Bcl-2 and Bax in the mitochondrial membrane.

Topics: Animals; Apoptosis; Caspases; Cell Death; Cells, Cultured; Cerebral Cortex; Disease Models, Animal; Embryo, Mammalian; Mitochondria; Nerve Degeneration; Neurotoxins; Nitro Compounds; Propionates; Rats; Rats, Wistar; Tacrolimus

The capacity of Schwann cells (SCs) in the peripheral nervous system to support axonal regeneration, in contrast to the oligodendrocytes in the central nervous system, has led to the misconception that peripheral nerve regeneration always restores function. Here, we consider how prolonged periods of time that injured neurons remain without targets during axonal regeneration (chronic axotomy) and that SCs in the distal nerve stumps remain chronically denervated (chronic denervation) progressively reduce the number of motoneurons that regenerate their axons. We demonstrate the effectiveness of low-dose, brain-derived neurotrophic and glial-derived neurotrophic factors to counteract the effects of chronic axotomy in promoting axonal regeneration. High-dose brain-derived neurotrophic factor (BDNF) on the other hand, acting through the p75 receptor, inhibits axonal regeneration and may be a factor in stopping regenerating axons from forming neuromuscular connections in skeletal muscle. The immunophilin, FK506, is also effective in promoting axonal regeneration after chronic axotomy. Chronic denervation of SCs (>1 month) severely deters axonal regeneration, although the few motor axons that do regenerate to reinnervate muscles become myelinated and form enlarged motor units in the reinnervated muscles. We found that in vitro incubation of chronically denervated SCs with transforming growth factor-beta re-established their growth-supportive phenotype in vivo, consistent with the idea that the interaction between invading macrophages and denervated SCs during Wallerian degeneration is essential to sustain axonal regeneration by promoting the growth-supportive SC phenotype. Finally, we consider the effectiveness of a brief period of 20 Hz electrical stimulation in promoting the regeneration of axons across the surgical gap after nerve repair.

Topics: Animals; Autonomic Denervation; Axons; Axotomy; Brain-Derived Neurotrophic Factor; Cell Count; Colforsin; Dextrans; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Interactions; Electric Stimulation; Evoked Potentials, Motor; Humans; In Vitro Techniques; Mice; Mice, Knockout; Motor Neurons; Muscle Contraction; Nerve Degeneration; Nerve Growth Factors; Nerve Regeneration; Peripheral Nerve Injuries; Peripheral Nerves; Rats; Receptor, Nerve Growth Factor; Receptor, trkB; Receptors, Nerve Growth Factor; Recovery of Function; Rhodamines; Schwann Cells; Tacrolimus; Time Factors; Transforming Growth Factor beta

A direct histological assay of axonal regeneration would have many advantages over currently available behavioral, electrophysiological, and radiometric assays. We show that peripheral sensory axons marked with the yellow fluorescent protein in transgenic mice can be viewed transcutaneously in superficial nerves. Degenerating and regenerating axons can be followed in live animals with a dissecting microscope and then, after fixation, studied at high resolution by confocal microscopy. Using this approach, we document differences in regenerative ability after nerve transection, crush injury, and crush injury after a previous "conditioning" lesion. We also show that the chemotherapeutic drug vincristine rapidly but transiently blocks regeneration and that the immunosuppressive drug FK506 modestly enhances regeneration. Moreover, FK506 nearly restores normal regenerative ability in animals treated with submaximal doses of vincristine. Because neuropathy is the major dose-limiting side effect of vincristine, we propose that its efficacy could be enhanced by coadministration of FK506 analogs that are neuroactive but not immunosuppressive.

Topics: Animals; Axons; Axotomy; Bacterial Proteins; Green Fluorescent Proteins; Kinetics; Luminescent Proteins; Mice; Mice, Transgenic; Microscopy, Fluorescence; Nerve Degeneration; Nerve Regeneration; Neuroprotective Agents; Neurotoxins; Peripheral Nerves; Tacrolimus; Vincristine

We show that nonimmunosuppressive analogues of the immunosuppressive drugs FK506 and cyclosporin A (CsA) rescue axotomized neonatal motor neuron death. Unilateral sciatic nerve was transected in neonatal rats. Animals were then treated daily with different doses of FK506 and CsA for 14 days with intraperitoneal injection. Control rats received phosphate buffer saline (PBS) in the same fashion. After treatment, the number of spinal motor neurons was determined at L4 level. In comparison with vehicle, both FK506 (5.0 mg kg(-1)) and CsA (10.0 mg kg(-1)) rescued motor neuron death in a similar way. These results indicate therapeutic relevance in the treatment of damaged motor neuron disorders, such as motor neuropathy or amyotrophic lateral sclerosis.

Topics: Animals; Animals, Newborn; Axotomy; Cell Death; Cyclosporine; Dose-Response Relationship, Drug; Motor Neurons; Nerve Degeneration; Neuroprotective Agents; Rats; Sciatic Nerve; Spinal Cord; Tacrolimus

The potential neuroprotective effects of the novel nitro-derivate of aspirin (NCX4016) on permanent focal cerebral ischemia in spontaneously hypertensive rats (SHRs) was investigated. Reference compounds were acetylsalicilic acid (ASA) and FK506 (tacrolimus). Ten minutes after surgery, SHRs were randomly divided into four groups of ten, pharmacologically treated and sacrificed 24 h after treatment. Brains were removed and processed to measure infarct volume, 70 kDa heat shock protein (hsp70), glial fibrillary acidic protein (GFAP) and vimentin (Vim) immunoreactivity (IR), and apoptosis using terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick end-labeling (TUNEL) assay. NCX-4016 significantly reduced total infarct volume compared to ASA (-20%, P < 0.05), FK506 (-18%, P < 0.05) and vehicle treatment (-20%, P < 0.05). Experimental groups did not differ in hsp70-IR and GFAP-IR. Conversely, hyperplastic astrocytes, measured by Vim-IR, were significantly lower in NCX-4016 than in the vehicle group (-36%, P<0.01). TUNEL assay indicated a significantly lower degree of apoptosis in NCX-4016 group than vehicle in both the homolateral (-27%, P < 0.01) and contralateral hemisphere (-29%, P < 0.05). These findings indicate that NO release associated with aspirin confers neuroprotective effects against ischemic injury.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Aspirin; Astrocytes; Brain; Brain Injuries; Brain Ischemia; Cell Survival; Functional Laterality; Glial Fibrillary Acidic Protein; HSP70 Heat-Shock Proteins; Immunohistochemistry; Immunosuppressive Agents; Male; Nerve Degeneration; Neurons; Neuroprotective Agents; Rats; Rats, Inbred SHR; Tacrolimus; Vimentin

Immunosuppressant drugs, like FK506, and nonimmunosuppressant compounds like, GPI1046 and L685818, are immunophilin ligands that specifically bind to immunophilins, like FK506 binding protein 12 (FKBP12). Several lines of evidence show that these ligands exert neurotrophic properties in neural injury models and in PC12 cells. However, the mechanism of the neurotrophic function of the immunophilin ligands is poorly known. In the present study, we use MPP+ and 6-OHDA toxicity models to examine both neuroprotective and neuroregenerative effects of immunophilin ligands on primary cultures of midbrain dopaminergic neurons. We find that FK506, GPI1046 and L685818 at concentrations from 0.01 to 1 microM partially, but significantly, protect dopaminergic neurons against both MPP+ and 6-OHDA toxicity. By Western blot analysis, we also find that all three compounds prevent tyrosine hydroxylase (TH) loss induced by MPP+ and 6-OHDA treatments. Morphologic analysis of dopaminergic neurons, by immunocytochemistry, shows that MPP+ and 6-OHDA cause the retraction and loss of neuronal processes, while FK506, GPI1046 and L685818 promote regeneration of these processes as indicated by increases in process number and length. To examine if FKBP12 is required for neurotrophic effects of immunophilin ligands, we cultured dopaminergic neurons from FKBP12 knockout mice and find that FK506 still protects dopaminergic neurons against MPP+ toxicity. These results suggest that FKBP12 is not essential for the neurotrophic properties of immunophilin ligands, and immunophilin ligands are a new class of neuroprotective and neuroregenerative agents that may have therapeutic potential in a variety of neurological disorders.

Topics: 1-Methyl-4-phenylpyridinium; Animals; Brain Injuries; Cells, Cultured; Dopamine; Immunophilins; Immunosuppressive Agents; Ligands; Mesencephalon; Mice; Mice, Knockout; Nerve Degeneration; Nerve Regeneration; Neurons; Neuroprotective Agents; Neurotoxins; Oxidopamine; Parkinsonian Disorders; Pyrrolidines; Rats; Rats, Sprague-Dawley; Tacrolimus; Tacrolimus Binding Protein 1A; Tyrosine 3-Monooxygenase

We examined the effects of the immunosuppressant tacrolimus (FK506) on the discrimination learning impairment induced by chronic cerebral hypoperfusion in rats. Chronic cerebral hypoperfusion was prepared by permanent ligation of bilateral common carotid arteries for male Wistar rats aged 9 weeks. FK506 (0.05 mg/kg, s.c.) recovered the learning impairment and also prevented the rarefaction of white matter and striatal neuronal cell damage. Our findings suggest that FK506 ameliorates the learning impairment mainly due to preventing neuropathological alterations.

Topics: Animals; Brain; Cerebrovascular Circulation; Chronic Disease; Corpus Callosum; Dementia, Vascular; Frontal Lobe; Hypoxia-Ischemia, Brain; Immunosuppressive Agents; Learning Disabilities; Male; Neostriatum; Nerve Degeneration; Nerve Fibers, Myelinated; Rats; Rats, Wistar; Recovery of Function; Tacrolimus

The immunosuppressant agent FK506 (tacrolimus) has proven to be neuroprotective against brain ischemia, but there are no data on potential neuroprotective effects of FK506 in peripheral nerve ischemia. We examined the potential effects of two doses of FK506 in protecting peripheral nerve from ischemic fiber degeneration. Ischemia in the left sciatic nerve of the rat was produced by injecting 2 x 10(6) microspheres (14 microm) into the left femoral, hypogastric, and superior gluteal arteries in proportions of 47%, 37%, and 17%, respectively. After embolization, FK506 was injected into the left femoral, hypogastric, and superior gluteal arteries in doses of 9.4, 7.4, and 3.4 microg, respectively, for the high-dose group and 4.7, 3.7, and 1.7 microg, respectively, for the low-dose group. The control rats were injected with saline. FK506 treatment resulted in dramatic behavioral improvement in nerve function, in the number of functioning nerve fibers, and in the salvage of a majority of nerve fibers from ischemic fiber degeneration in a dose-dependent fashion. These results suggest that a small dose of FK506 protects peripheral nerve from ischemic fiber degeneration and that it may have potential in the treatment of ischemic neuropathy.

Topics: Action Potentials; Animals; Behavior, Animal; Dose-Response Relationship, Drug; Electrophysiology; Immunosuppressive Agents; Ischemia; Male; Nerve Degeneration; Nerve Fibers; Rats; Rats, Sprague-Dawley; Regional Blood Flow; Tacrolimus; Tibial Nerve

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.

Topics: Animals; Axons; Immunosuppressive Agents; Male; Microscopy, Electron; Nerve Degeneration; Nerve Regeneration; Neural Pathways; Neurons; Peripheral Nerves; Rats; Rats, Sprague-Dawley; Red Nucleus; Spinal Cord; Spinal Cord Injuries; Tacrolimus

To evaluate the effect of FK506 on delayed neuronal death in gerbils after forebrain ischemia, 84 adult Mongolian gerbils were used in this study. Transient forebrain ischemia was induced by clipping common carotid arteries bilaterally for 5 minutes. One hour after reperfusion we intraperitoneally injected FK506 (1.0 mg/kg), cyclosporin A (CsA) (10.0 mg/kg) or the vehicle solution into each gerbil. In one group, each agent was additionally administered daily 3 more times at 24, 48 and 72 hours after ischemia. The gerbils were killed 4 days or 10 days after transient ischemia, and damage to their hippocampal pyramidal cells was histologically assessed. Additionally, the body temperature was measured following administration of each drug to investigate drug-induced hypothermia. Post-ischemic repeated treatment with FK506 significantly (p < 0.01) reduced degeneration of hippocampal neurons. However, partial treatment did not modify neuronal degeneration. CsA did not show a neuroprotective effect in this study. Drug-induced mild hypothermia (35-37 C) was observed following administration of FK506 or CsA. There was no significant difference in the time course of the body temperature between the FK506 and CsA group. We demonstrated that the repeated FK506 treatment, but not the CsA treatment, reduced ischemia-induced degeneration of hippocampal neurons in gerbils. Although FK506-induced hypothermia might have modified neuronal degeneration, a comparison with CsA indicated that the neuroprotective effect of FK506 was not solely due to hypothermia per se.

Topics: Animals; Body Temperature; Cell Death; Cyclosporine; Female; Gerbillinae; Hippocampus; Immunosuppressive Agents; Ischemic Attack, Transient; Male; Nerve Degeneration; Neuroprotective Agents; Pyramidal Cells; Tacrolimus

Nitric oxide (NO) is a potent biological messenger molecule in the central nervous system (CNS). There are several potential sources of NO production in the CNS, including neurons and endothelial cells which express NO synthase (NOS) constitutively. Astrocytes and microglia can be induced by cytokines to express a NOS isoform similar to macrophage NOS (mNOS). Primary mixed glial cultures exposed to lipopolysaccharide (LPS) or a combination of LPS and gamma-interferon (INF-gamma) produce nitrite, a breakdown product of NO formation, in a dose-dependent manner. Nitrite production is detectable at 12 hr, peaks at 48 hr and is sustained for at least 96 hr. The NOS inhibitor, nitro-L-arginine (NArg), inhibits nitrite formation, but the immunosuppressant agent, FK506, does not. In mixed glial-neuronal cultures exposed to 50 ng LPS or 5 ng LPS and 1 microgram INF-gamma, neurons begin to die at 48 hr, approx. 24-36 hr after detectable nitrite production. Neurotoxicity is attenuated by 100 microM NArg. These data indicate that expression of inducible mNOS causes delayed neurotoxicity.

Topics: Amino Acid Oxidoreductases; Animals; Arginine; Cells, Cultured; Cerebral Cortex; Interferon-gamma; Lipopolysaccharides; Nerve Degeneration; Nervous System Diseases; Neuroglia; Neurons; Nitric Oxide Synthase; Nitrites; Nitroarginine; Rats; Tacrolimus