Nitric oxide prevents axonal degeneration by inducing HIF-1-dependent expression of erythropoietin.

Nitric oxide (NO) is a signaling molecule that can trigger adaptive (physiological) or maladaptive (pathological) responses to stress stimuli in a context-dependent manner. We have previously reported that NO may signal axonal injury to neighboring glial cells. In this study, we show that mice deficient in neuronal nitric oxide synthase (nNOS-/-) are more vulnerable than WT mice to toxin-induced peripheral neuropathy. The administration of NO donors to primary dorsal root ganglion cultures prevents axonal degeneration induced by acrylamide in a dose-dependent manner. We demonstrate that NO-induced axonal protection is dependent on hypoxia-inducible factor (HIF)-1-mediated transcription of erythropoietin (EPO) within glial (Schwann) cells present in the cultures. Transduction of Schwann cells with adenovirus AdCA5 encoding a constitutively active form of HIF-1α results in amelioration of acrylamide-induced axonal degeneration in an EPO-dependent manner. Mice that are partially deficient in HIF-1α (HIF-1α+/-) are also more susceptible than WT littermates to toxic neuropathy. Our results indicate that NO→HIF-1→EPO signaling represents an adaptive mechanism that protects against axonal degeneration.

Axonal protective effects of the myelin-associated glycoprotein.

Progressive axonal degeneration follows demyelination in many neurological diseases, including multiple sclerosis and inherited demyelinating neuropathies, such as Charcot-Marie-Tooth disease. One glial molecule, the myelin-associated glycoprotein (MAG), located in the adaxonal plasmalemma of myelin-producing cells, is known to signal to the axon and to modulate axonal caliber through phosphorylation of axonal neurofilament proteins. This report establishes for the first time that MAG also promotes resistance to axonal injury and prevents axonal degeneration both in cell culture and in vivo. This effect on axonal stability depends on the RGD domain around arginine 118 in the extracellular portion of MAG, but it is independent of Nogo signaling in the axon. Exploiting this pathway may lead to therapeutic strategies for neurological diseases characterized by axonal loss.

Establishment of a rodent model of HIV-associated sensory neuropathy.

Human immunodeficiency virus (HIV)-associated sensory neuropathy (SN) is the most common neurological complication of HIV infection in the current highly active antiretroviral therapy era. The painful sensory neuropathy is associated with the use of dideoxynucleoside antiretrovirals, and its development limits the choice of antiretroviral drugs in affected patients. There are presently no effective therapies for HIV-SN, and moreover there has been no robust animal model of HIV-SN in which candidate therapeutic agents can be tested. In this paper, we show that we have established a rodent model of HIV-SN by oral administration of a dideoxynucleoside drug, didanosine, to transgenic mice expressing the HIV coat protein gp120 under a GFAP promoter. The neuropathy in these rodents is characterized by distal degeneration of unmyelinated sensory axons, similar to the "dying back" pattern of C-fiber loss seen in patients with HIV-SN. This model will be useful in examining mechanisms of distal axonal degeneration and testing potential neuroprotective compounds that may prevent development of the sensory neuropathy.

Demyelinating disorders: update on transverse myelitis.

Transverse myelitis (TM) is a focal inflammatory disorder of the spinal cord. Perivascular monocytic and lymphocytic infiltration, demyelination, and axonal injury are prominent histopathogic features of TM. The clinical manifestations of TM are consequent to dysfunction of motor, sensory, and autonomic pathways. At peak deficit, 50% of patients with TM are completely paraplegic (with no volitional movements of legs), virtually all have some degree of bladder dysfunction, and 80% to 94% have numbness, paresthesias, or band-like dysesthesias. Longitudinal case series of TM reveal that approximately one third of patients recover with little to no sequelae, one third are left with a moderate degree of permanent disability, and one third have severe disability. Recent studies have shown that the cytokine interleukin-6 may be a useful biomarker, as the levels of interleukin-6 in the cerebrospinal fluid of acute TM patients strongly correlate with and are highly predictive of disability. Clinical trials testing the efficacy of promising axonoprotective agents in combination with intravenous steroids in the treatment of TM are currently underway.

Spatially distinct and functionally independent mechanisms of axonal degeneration in a model of HIV-associated sensory neuropathy.

Sensory polyneuropathies are the most frequent neurological complication of human immunodeficiency virus (HIV) infection. Distal symmetric polyneuropathy (DSP), associated with HIV infection, is characterized by length-dependent axonal degeneration of sensory fibres. In rodent dorsal root ganglia (DRG) cultures, HIV viral envelope protein gp120 results in neurotoxicity and axonal degeneration. Since it is unknown whether the axonal degeneration is a consequence of neuronal death or whether it is due to a direct toxic effect on axons, we investigated gp120-induced axonal toxicity using compartmentalized cultures of sensory neurons. Our results show that gp120 causes neuronal apoptosis and axonal degeneration through two, independent and spatially separated mechanisms of action: (i) an indirect insult to cell bodies, requiring the presence of Schwann cells, results in neuronal apoptotic death and subsequent axonal degeneration; (ii) a direct, local toxicity exerted on axons through activation of mitochondrial caspase pathway that is independent of cell body. This local axonal toxicity is mediated through binding of gp120 to axonal chemokine receptors and can be prevented by chemokine receptor blockers. In conclusion, we propose a novel pathway of axonal degeneration mediated by gp120 that is dependent on local activation of caspases in the axon. This observation suggests that axonal protection is a relevant therapeutic target for HIV-associated sensory neuropathy. Furthermore, chemokine receptor inhibitors, which are currently being developed as HIV entry inhibitor drugs, may also have a therapeutic role in HIV-associated peripheral neuropathies by preventing axonal degeneration.

IL-6 induces regionally selective spinal cord injury in patients with the neuroinflammatory disorder transverse myelitis.

Transverse myelitis (TM) is an immune-mediated spinal cord disorder associated with inflammation, demyelination, and axonal damage. We investigated the soluble immune derangements present in TM patients and found that IL-6 levels were selectively and dramatically elevated in the cerebrospinal fluid and directly correlated with markers of tissue injury and sustained clinical disability. IL-6 was necessary and sufficient to mediate cellular injury in spinal cord organotypic tissue culture sections through activation of the JAK/STAT pathway, resulting in increased activity of iNOS and poly(ADP-ribose) polymerase (PARP). Rats intrathecally infused with IL-6 developed progressive weakness and spinal cord inflammation, demyelination, and axonal damage, which were blocked by PARP inhibition. Addition of IL-6 to brain organotypic cultures or into the cerebral ventricles of adult rats did not activate the JAK/STAT pathway, which is potentially due to increased expression of soluble IL-6 receptor in the brain relative to the spinal cord that may antagonize IL-6 signaling in this context. The spatially distinct responses to IL-6 may underlie regional vulnerability of different parts of the CNS to inflammatory injury. The elucidation of this pathway identifies specific therapeutic targets in the management of CNS autoimmune conditions.

Neuroprotection in the PNS: erythropoietin and immunophilin ligands.

Many illnesses that affect the peripheral nervous system (PNS) lead to distal axonal degeneration rather than loss of neuronal cell bodies. Strategies aimed at promoting survival of injured neurons (i.e., preventing cell death) may not be applicable to many PNS illnesses. We have developed in vitro and in vivo animal models to study mechanisms of acquired peripheral neuropathies and used these models to evaluate the therapeutic potential of novel compounds. In recent years, erythropoietin (EPO) has been recognized as a novel neuroprotectant in the central nervous system. In the PNS, we recently showed that Schwann cell-derived EPO acts as an endogenous neuroprotectant and that it is most effective in preventing distal axonal degeneration seen in models of peripheral neuropathy. Similarly, we showed that immunophilin ligands are also neuroprotective in the PNS and prevent axonal degeneration seen in models of peripheral neuropathies. Both EPO and non-immunosuppressive immunophilin ligands are in early clinical development for the treatment of acquired peripheral neuropathies.

Etiology of perfusion-diffusion magnetic resonance imaging mismatch patterns.

BACKGROUND AND PURPOSE: Diffusion-and perfusion-weighted magnetic resonance imaging (DWI and PWI) are useful tools for the assessment of brain ischemia. Discrepancies between the extent of DWI and PWI abnormalities are thought to depend pre dominantly on time from symptom onset to magnetic resonance imaging (MRI) examination. However, underlying ischemic stroke etiology can also be important. A mismatch may indicate the presence of tissue at risk for infarction, whereas the relevance of other DWI/PWI patterns is uncertain. The authors therefore investigated the etiology of brain ischemia in patients with different DWI/PWI patterns. METHODS: Retrospective study of 130 patients with acute brain ischemia and detailed stroke workup, including MRI within a week after symptom onset (40 +/- 39 hours). Patients were divided into the following groups: mis-match (PWI > DWI), reverse mismatch (DWI > PWI), and match (<25% difference between PWI and DWI). RESULTS: Mismatch occurred in 49% of patients, whereas 22% had reverse mis-match and 29% matched lesions. Time from symptom onset to MRI examination was similar between the 3 groups. Largeartery atherosclerosis increased by almost 4-fold the odds of mismatch (odds ratio: 3.89, 95% confidence interval: 1.72-8.78; P < .001), whereas patients with reverse mismatch were likely to have cryptogenic stroke. Patients with matched lesions were similarly distributed among different stroke subtypes. CONCLUSIONS: Ischemic stroke etiology appears to influence the development of specific DWI/PWI patterns. Prospective studies are needed to confirm these observations.

Erythropoietin is neuroprotective in models of HIV sensory neuropathy.

HIV-associated sensory neuropathy (HIV-SN) is the most common neurological complication of HIV infection. Presently, there are no effective therapies for this painful neuropathy. The pathology of HIV-SN is characterized by 'dying back' sensory axonal degeneration and a more modest loss of dorsal root ganglion (DRG) sensory neurons. It has been hypothesized that HIV-SN results from neurotoxicity by secreted viral proteins, such as the HIV envelope glycoprotein gp120. Furthermore, neurotoxicity by dideoxynucleoside (DDX) agents, results in the observed higher incidence of HIV-SN in HIV-infected patients taking these antiretroviral drugs. In this study we show that administration of picomolar amounts of the hormone erythropoietin (EPO) prevents sensory axonal degeneration and in vitro DRG neuronal death by both gp120 and ddC (a neurotoxic DDX drug). Our results suggest that EPO may be useful in the treatment of HIV-SN.

A novel endogenous erythropoietin mediated pathway prevents axonal degeneration.

Clinically relevant peripheral neuropathies (such as diabetic and human immunodeficiency virus sensory neuropathies) are characterized by distal axonal degeneration, rather than neuronal death. Here, we describe a novel, endogenous pathway that prevents axonal degeneration. We show that in response to axonal injury, periaxonal Schwann cells release erythropoietin (EPO), which via EPO receptor binding on neurons, prevents axonal degeneration. We demonstrate that the relevant axonal injury signal that stimulates EPO production from surrounding glial cells is nitric oxide. In addition, we show that this endogenous pathway can be therapeutically exploited by administering exogenous EPO. In an animal model of distal axonopathy, systemic EPO administration prevents axonal degeneration, and this is associated with a reduction in limb weakness and neuropathic pain behavior. Our in vivo and in vitro data suggest that EPO prevents axonal degeneration and therefore may be therapeutically useful in a wide variety of human neurological diseases characterized by axonopathy.

Clinical and genetic description of a family with Charcot-Marie-Tooth disease type 1B from a transmembrane MPZ mutation.

Mutations in the myelin protein zero gene (MPZ) are associated with certain demyelinating neuropathies, and in particular with Charcot-Marie-Tooth disease type 1B (CMT1B), Dejerine-Sottas syndrome, and congenital hypomyelination. MPZ mutations affecting the protein's transmembrane domain are generally associated with more severe phenotypes. We describe a family with mild CMT1B associated with a transmembrane MPZ mutation. Sequence analysis identified a G-to-C transversion at nucleotide 1064, predicting a glycine-to-arginine substitution in codon 163 (G163R) of MPZ. This report furthers the understanding of the clinical and electrophysiological manifestations of MPZ mutations.

The use of GAP-43 mRNA quantification in high throughput screening of putative neuroprotective agents in dorsal root ganglion cultures.

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.

Schwann cell chemokine receptors mediate HIV-1 gp120 toxicity to sensory neurons.

Human immunodeficiency virus (HIV)-associated sensory neuropathy (HIV-SN) is the most common neurological complication of HIV infection. Currently, the pathogenesis of HIV-SN is unknown. Because there is no convincing evidence of neuronal infection, HIV neurotoxicity is likely to be effected either by secreted viral proteins such as the envelope glycoprotein gp120 or by neurotoxic cytokines released from infected/activated glial cells. We describe a model of gp120 toxicity to primary sensory neurons, in which gp120 induces neuritic degeneration and neuronal apoptosis. We show that Schwann cells, the cells that ensheath peripheral nerve axons, and which traditionally have been viewed as having a passive, supporting role, mediate this neurotoxicity. Ligation of the chemokine receptor CXCR4 on Schwann cells by gp120 resulted in the release of RANTES, which induced dorsal root ganglion neurons to produce tumor necrosis factor-alpha and subsequent TNFR1-mediated neurotoxicity in an autocrine fashion. This newly described Schwann cell-neuron interaction may be pathogenically relevant not only in HIV-SN but also in other peripheral neuropathies.

FK506 is neuroprotective in a model of antiretroviral toxic neuropathy.

Antiretroviral toxic neuropathy is the most common neurological complication of human immunodeficiency virus infection. This painful neuropathy not only affects the quality of life of human immunodeficiency virus-infected patients but also severely limits viral suppression strategies. We have developed an in vitro model of this toxic neuropathy to better understand the mechanism of neurotoxicity and to test potential neuroprotective compounds. We show that among the dideoxynucleosides, ddC appears to be the most neurotoxic, followed by ddI and then d4T. This reflects their potency in causing neuropathy. AZT, which does not cause a peripheral neuropathy in patients, does not cause significant neurotoxicity in our model. Furthermore, in this model, we show that the immunophilin ligand FK506 but not cyclosporin A prevents the development of neurotoxicity by ddC, as judged by amelioration of ddC-induced "neuritic pruning," neuronal mitochondrial depolarization, and neuronal necrotic death. This finding suggests a calcineurin-independent mechanism of neuroprotection. As calcineurin inhibition underlies the immunosuppressive properties of these clinically used immunophilin ligands, this holds promise for the neuroprotective efficacy of nonimmunosuppressive analogs of FK506 in the prevention or treatment of antiretroviral toxic neuropathy.