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.

Pharmacokinetic and Target Engagement Measures of ANX007, an Anti-C1q Antibody Fragment, Following Intravitreal Administration in Nonhuman Primates.

Purpose: C1q and the classical complement cascade are key regulators of synaptic pruning, and their aberrant activation has been implicated in neurodegenerative ophthalmic diseases including geographic atrophy and glaucoma. The antigen-binding fragment antibody ANX007 specifically recognizes globular head groups of C1q to block substrate binding and functionally inhibit classical complement cascade activation. ANX007 was assessed in nonclinical studies of biodistribution and C1q target engagement in the eye following intravitreal (IVT) administration in cynomolgus monkeys. Methods: Female juvenile cynomolgus monkeys (n = 12) received a single bilateral dose of 1 or 5 mg ANX007/eye, with vitreous and non-perfused tissue samples collected approximately 4 weeks later. In a separate study, male (n = 6/5) and female (n = 6/5) animals received repeat bilateral dosing of 1, 2.5, or 5 mg ANX007/eye on days 1 and 29, with aqueous and vitreous collections on day 44 or day 59. Tissues from the 5 mg/eye repeat-dose group were perfused, and retina, choroid, and optic nerve samples were collected approximately 2 and 4 weeks post-last dose. Results: Following a single dose of ANX007, vitreous levels of free drug were measurable through 4 weeks at both the 1 and 5 mg dose levels, with approximately 3-day half-life. With repeat dose of 5 mg/eye, free-ANX007 was measurable 4 weeks post-last dose in perfused retina and choroid and up to approximately 2 weeks post-last dose in optic nerve. There was a strong correlation between C1q target engagement and free drug levels in aqueous and vitreous humors and retinal tissue. Conclusions: Following IVT administration, ANX007 distributes to sites within the retina that are relevant to neurodegenerative ophthalmic disease with clear evidence of C1q target engagement. Based on its mechanism of action inhibiting C1q and its downstream activity, ANX007 is predicted to mitigate tissue damage driven by classical complement activation in the retina. These data support further clinical evaluation of ANX007.

Proof-of-Mechanism Study of the Phosphodiesterase 10 Inhibitor RG7203 in Patients With Schizophrenia and Negative Symptoms.

Background: Reduced activation of dopamine D1 receptor signaling may be implicated in reward functioning as a potential driver of negative symptoms in schizophrenia. Phosphodiesterase 10A (PDE10A), an enzyme that is highly expressed in the striatum, modulates both dopamine D2- and D1-dependent signaling. Methods: We assessed whether augmentation of D1 signaling by the PDE10 inhibitor RG7203 enhances imaging and behavioral markers of reward functions in patients with schizophrenia and negative symptoms. In a 3-period, double-blind, crossover study, we investigated the effects of RG7203 (5 mg and 15 mg doses) and placebo as adjunctive treatment to stable background antipsychotic treatment in patients with chronic schizophrenia with moderate levels of negative symptoms. Effects on reward functioning and reward-based effortful behavior were evaluated using the monetary incentive delay task during functional magnetic resonance imaging and the effort-cost-benefit and working memory reinforcement learning tasks. Results: Patients (N = 33; 30 male, mean age ± SD 36.6 ± 7.0 years; Positive and Negative Syndrome Scale negative symptom factor score 23.0 ± 3.5 at screening) were assessed at three study centers in the United States; 24 patients completed the study. RG7203 at 5 mg significantly increased reward expectation-related activity in the monetary incentive delay task, but in the context of significantly decreased overall activity across all task conditions. Conclusions: In contrast to our expectations, RG7203 significantly worsened reward-based effortful behavior and indices of reward learning. The results do not support the utility of RG7203 as adjunctive treatment for negative symptoms in patients with schizophrenia.

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.

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.

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.

Granzyme B mediates neurotoxicity through a G-protein-coupled receptor.

Neuroinflammatory diseases such as multiple sclerosis (MS) are characterized by focal regions of demyelination and axonal loss associated with infiltrating T cells. However, the role of activated T cells in causing neuronal injury remains unclear. CD4 and CD8 T cells were isolated from normal donors and polyclonally activated using plate-bound anti-CD3 and soluble anti-CD28. The conditioned T cell supernatants caused toxicity to cultured human fetal neurons, which could be blocked by immunodepleting the supernatants of granzyme B (GrB). Recombinant GrB also caused toxicity in neurons by caspase-dependent pathways but no toxicity was seen in astrocytes. The neurotoxicity was independent of perforin and could not be blocked by mannose-6-phosphate. However, GrB-induced neurotoxicity was sensitive to pertussis toxin, implicating the stimulation of Gialpha protein-coupled receptors. GrB caused a decrease in cAMP levels but only modest increases in intracellular calcium. The effect on intracellular calcium could be markedly potentiated by stromal-derived factor 1alpha. GrB-induced neurotoxicity could also be blocked by vitamin E and a neuroimmunophilin ligand. In conclusion, GrB may be an important mediator of neuronal injury in T cell-mediated neuroinflammatory disorders.

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.

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.

Safety, pharmacokinetic, and positron emission tomography evaluation of serotonin and dopamine transporter occupancy following multiple-dose administration of the triple monoamine reuptake inhibitor BMS-820836.

RATIONALE: BMS-820836 is a novel antidepressant that selectively inhibits the reuptake of serotonin, norepinephrine, and dopamine. OBJECTIVE: This Phase I study assessed safety, tolerability, and pharmacokinetics of multiple daily doses of BMS-820836 in healthy subjects. Central serotonin transporter (SERT) and dopamine transporter (DAT) occupancy were assessed using positron emission tomography and [(11)C]MADAM or [(11)C]PE2I, respectively. METHODS: Fifty-seven healthy volunteers were enrolled in this double-blind, placebo-controlled, ascending multiple-dose study (ClincalTrials.gov identifier: NCT00892840). Eight participants in seven dose cohorts received oral doses of BMS-820836 (0.1-4 mg) or placebo for 14 days to assess safety, tolerability, and pharmacokinetics. Additionally, SERT and DAT occupancies were evaluated in 4-8 subjects per cohort at 8 h post-dose on Day 10 and 24 h post-dose on Day 15 at anticipated steady-state conditions. RESULTS: Most adverse events were mild to moderate; there were no serious safety concerns. Median maximum concentrations of BMS-820836 were observed at 4.0-5.5 h post-dose; estimated elimination half-life was 44-74 h. About 80 % striatal SERT occupancy was achieved after multiple doses of 0.5 mg BMS-820836 at both 8 and 24 h post-dose. Striatal DAT occupancy ranged between 14 % and 35 % at 8 h post-dose with a slight decline at 24 h post-dose. CONCLUSIONS: Multiple daily doses of up to 4 mg BMS-820836 appeared to be generally safe and well tolerated in a healthy population. SERT and DAT occupancies were in a range associated with therapeutic efficacy of antidepressants. Together with the pharmacokinetic profile of BMS-820836, the occupancy data support once-daily administration.

Comparison of different QT interval correction methods for heart rate and QT beat-to-beat method in a thorough QT study of triple monoamine reuptake inhibitor BMS-820836.

As BMS-820836 causes dose-dependent heart rate increases, QTcI, QTcF, QTcB, and QT beat-to-beat methods were compared in this thorough QT study in healthy subjects. Two parallel groups of subjects (n = 60 per group) received 2 mg (maximum therapeutic) or 4 mg (supratherapeutic) of BMS-820836 once daily for 14 days with uptitration. Another 60 subjects received encapsulated moxifloxacin (400 mg) or matching placebo in a nested-crossover study design. Compared with QTcF and QTcB, baseline QTcI had the smallest and near-zero Pearson correlation coefficient with heart rate (0.0938), which supported the choice of QTcI as the primary electrocardiographic end point. BMS-820836 was not associated with prolongation of the QT interval at the doses tested; however, ΔΔQTbtb showed the smallest deviation from 0 milliseconds compared with ΔΔQTcI and ΔΔQTcF. The ΔΔQTbtb results appeared to be more consistent with the very low likelihood of any effect on the QT interval by BMS-820836 based on the wide margin (>400×) of the in vitro hERG IC50 and free plasma concentration. Further, this is the first published study that used the nested-crossover design and QTbtb analysis in a thorough QT study. Assay sensitivity was confirmed with encapsulated moxifloxacin.

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.

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.

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.

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.