Search

IntroductionAnimal models of neuromyelitis optica (NMO) are needed for drug testing and evaluation of NMO disease pathogenesis mechanisms.ResultsWe describe a novel passive-transfer model of NMO in which rats made seropositive for human anti-aquaporin-4 (AQP4) immunoglobulin G antibody (NMO-IgG) by intraperitoneal (IP) injections were subject to intracerebral needle injury. Following a single IP injection, NMO-IgG distributed rapidly to peripheral AQP4-expressing cells (kidney collecting duct, gastric glands, airways, skeletal muscle) and area postrema in brain, but not elsewhere in the central nervous system; however, no pathology was seen in brain, spinal cord, optic nerve or peripheral tissues. After testing various maneuvers to produce NMO-IgG-dependent pathology in brain, we found that transient puncture of brain parenchyma with a 28-gauge needle in NMO-IgG seropositive rats produced robust NMO pathology around the needle track, with loss of AQP4 and glial fibrillary acidic protein, granulocyte and macrophage infiltration, centrovascular deposition of activated complement, and blood-brain barrier disruption, with demyelination by 5 days. Pathology was not seen in rats receiving control (non-NMO) human IgG or in NMO-IgG-seropositive rats made complement-deficient by cobra venom factor. Interestingly, at 1 day a reversible, multifocal astrocytopathy was seen with loss of AQP4 and GFAP (but not myelin) in areas away from the needle track.ConclusionsNMO-IgG-seropositivity alone is not sufficient to cause NMO pathology in rats, but a single intracerebral needle insertion, without pre-existing inflammation or infusion of pro-inflammatory factors, was sufficient to produce robust NMO pathology in seropositive rats.

Astrocyte water channel aquaporin-4 (AQP4) facilitates water movement across the blood-brain barrier and into injured astrocytes. We previously showed reduced cytotoxic brain edema with improved neurological outcome in AQP4 knockout mice in water intoxication, infection and cerebral ischemia. Here, we established a 4-vessel transient occlusion model to test the hypothesis that AQP4 deficiency in mice could improve neurological outcome following severe global cerebral ischemia as occurs in cardiac arrest/resuscitation. Mice were subjected to 10-min transient bilateral carotid artery occlusion at 24h after bilateral vertebral artery cauterization. Cerebral blood flow was reduced during occlusion by >94% in both AQP4(+/+) and AQP4(-/-) mice. The primary outcome, neurological score, was remarkably better at 3 and 5 days after occlusion in AQP4(-/-) than in AQP4(+/+) mice, and survival was significantly improved as well. Brain water content was increased by 2.8±0.4% in occluded AQP4(+/+) mice, significantly greater than that of 0.3±0.6% in AQP4(-/-) mice. Histological examination and immunofluorescence of hippocampal sections at 5 days showed significantly greater neuronal loss in the CA1 region of hippocampus in AQP4(+/+) than AQP4(-/-) mice. The neuroprotection in mice conferred by AQP4 deletion following severe global cerebral ischemia provides proof-of-concept for therapeutic AQP4 inhibition to improve neurological outcome in cardiac arrest.

Animal models of neuromyelitis optica (NMO) are needed for elucidation of disease mechanisms and for testing new therapeutics. Prior animal models of NMO involved administration of human anti-aquaporin-4 immunoglobulin G antibody (NMO-IgG) to rats with pre-existing neuroinflammation, or to naïve mice supplemented with human complement. We report here the development of NMO pathology following passive transfer of NMO-IgG to naïve rats. A single intracerebral infusion of NMO-IgG to adult Lewis rats produced robust lesions around the needle track in 100 % of rats; at 5 days there was marked loss of aquaporin-4 (AQP4), glial fibrillary acidic protein (GFAP) and myelin, granulocyte and macrophage infiltration, vasculocentric complement deposition, blood-brain barrier disruption, microglial activation and neuron death. Remarkably, a distinct 'penumbra' was seen around lesions, with loss of AQP4 but not of GFAP or myelin. No lesions or penumbra were seen in rats receiving control IgG. The size of the main lesion with loss of myelin was greatly reduced in rats made complement-deficient by cobra venom factor or administered NMO-IgG lacking complement-dependent cytotoxicity (CDC) effector function. However, the penumbra was seen under these conditions, suggesting a complement-independent pathogenesis mechanism. The penumbra was absent with NMO-IgG lacking both CDC and antibody-dependent cellular cytotoxicity (ADCC) effector functions. Finally, lesion size was significantly reduced after macrophage depletion with clodronate liposomes. These results: (i) establish a robust, passive-transfer model of NMO in rats that does not require pre-existing neuroinflammation or complement administration; (ii) implicate ADCC as responsible for a unique type of pathology also seen in human NMO; and (iii) support a pathogenic role of macrophages in NMO.

The water channel aquaporin-4 (AQP4) is the target of the immunoglobulin G autoantibody (AQP4-IgG) in neuromyelitis optica (NMO). AQP4 is expressed in foot processes of astrocytes throughout the central nervous system, as well as in skeletal muscle and epithelial cells in kidney, lung and gastrointestinal organs. Phenotype analysis of AQP4 knockout mice indicates the involvement of AQP4 in water movement into and out of the brain, astrocyte migration, glial scar formation and neuroexcitatory phenomena. AQP4 monomers form tetramers in membranes, which further aggregate to form supramolecular assemblies called orthogonal arrays of particles. AQP4-IgG is pathogenic in NMO by a mechanism involving complement- and cell-mediated astrocyte cytotoxicity, which produces an inflammatory response with oligodendrocyte injury and demyelination. AQP4 orthogonal arrays are crucial in NMO pathogenesis, as they increase AQP4-IgG binding to AQP4 and greatly enhance complement-dependent cytotoxicity. Novel NMO therapeutics are under development that target AQP4-IgG or AQP4, including aquaporumab monoclonal antibodies and small molecules that block AQP4-IgG binding to AQP4, and enzymatic inactivation strategies to neutralize AQP4-IgG pathogenicity.

Neuromyelitis optica (NMO) is an inflammatory demyelinating disease of the central nervous system that can cause paralysis and blindness. The pathogenesis of NMO involves binding of immunoglobulin G autoantibodies to aquaporin-4 (AQP4) on astrocytes, which is thought to cause complement-dependent cytotoxicity (CDC) and a secondary inflammatory response leading to oligodendrocyte and neuronal damage. Here, we investigate in vivo the role of antibody-dependent cell-mediated cytotoxicity (ADCC) triggered by AQP4 autoantibodies (AQP4-IgG) in the development of NMO pathology. A high-affinity, human recombinant monoclonal AQP4-IgG was mutated in its Fc region to produce 'NMO superantibodies' with enhanced CDC and/or ADCC effector functions, without altered AQP4 binding. Pathological effects of these antibodies were studied in a mouse model of NMO produced by intracerebral injection of AQP4-IgG and human complement. The original (non-mutated) antibody produced large NMO lesions in this model, with loss of AQP4 and GFAP immunoreactivity, inflammation and demyelination, as did a mutated antibody with enhanced CDC and ADCC effector functions. As anticipated, a mutated AQP4-IgG lacking CDC, but having tenfold enhanced ADCC, produced little pathology. However, unexpectedly, a mutated antibody with ninefold enhanced CDC, but lacking ADCC, produced much less pathology than the original AQP4-IgG. Also, pathology was greatly reduced following administration of AQP4-IgG and complement to mice lacking the FcγIII receptor involved in effector cell activation during ADCC, and to normal mice injected with an Fcγ receptor blocking antibody. Our results provide evidence for the central involvement of ADCC in NMO pathology and suggest ADCC as a new therapeutic target in NMO.

Neuromyelitis optica (NMO) is an inflammatory demyelinating disease of the central nervous system caused by binding of pathogenic IgG autoantibodies (NMO-IgG) to astrocyte water channel aquaporin-4 (AQP4). Astrocyte damage and downstream inflammation require NMO-IgG effector function to initiate complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC). Here, we evaluated the potential therapeutic utility of the bacterial enzyme IdeS (IgG-degrading enzyme of Streptococcus pyogenes), which selectively cleaves IgG antibodies to yield Fc and F(ab')(2) fragments. In AQP4-expressing cell cultures, IdeS treatment of monoclonal NMO-IgGs and NMO patient sera abolished CDC and ADCC, even when IdeS was added after NMO-IgG was bound to AQP4. Binding of NMO-IgG to AQP4 was similar to that of the NMO-F(ab')(2) generated by IdeS cleavage. NMO-F(ab')(2) competitively displaced pathogenic NMO-IgG, preventing cytotoxicity, and the Fc fragments generated by IdeS cleavage reduced CDC and ADCC. IdeS efficiently cleaved NMO-IgG in mice in vivo, and greatly reduced NMO lesions in mice administered NMO-IgG and human complement. IgG-selective cleavage by IdeS thus neutralizes NMO-IgG pathogenicity, and yields therapeutic F(ab')(2) and Fc fragments. IdeS treatment, by therapeutic apheresis or direct administration, may be beneficial in NMO.

Neuromyelitis optica (NMO) is an autoimmune disorder with inflammatory demyelinating lesions in the central nervous system, particularly in the spinal cord and optic nerve. NMO pathogenesis is thought to involve binding of anti-aquaporin-4 (AQP4) autoantibodies to astrocytes, which causes complement-dependent cytotoxicity (CDC) and downstream inflammation leading to oligodendrocyte and neuronal injury. Vasculocentric deposition of activated complement is a prominent feature of NMO pathology. Here, we show that a neutralizing monoclonal antibody against the C1q protein in the classical complement pathway prevents AQP4 autoantibody-dependent CDC in cell cultures and NMO lesions in ex vivo spinal cord slice cultures and in mice. A monoclonal antibody against human C1q with 11 nM binding affinity prevented CDC caused by NMO patient serum in AQP4-transfected cells and primary astrocyte cultures, and prevented complement-dependent cell-mediated cytotoxicity (CDCC) produced by natural killer cells. The anti-C1q antibody prevented astrocyte damage and demyelination in mouse spinal cord slice cultures exposed to AQP4 autoantibody and human complement. In a mouse model of NMO produced by intracerebral injection of AQP4 autoantibody and human complement, the inflammatory demyelinating lesions were greatly reduced by intracerebral administration of the anti-C1q antibody. These results provide proof-of-concept for C1q-targeted monoclonal antibody therapy in NMO. Targeting of C1q inhibits the classical complement pathway directly and causes secondary inhibition of CDCC and the alternative complement pathway. As C1q-targeted therapy leaves the lectin complement activation pathway largely intact, its side-effect profile is predicted to differ from that of therapies targeting downstream complement proteins.

Neuronal hyperactivity is the emerging functional hallmark of Alzheimer's disease (AD) in both humans and different mouse models, mediating an impairment of memory and cognition. The mechanisms underlying neuronal hyperactivity remain, however, elusive. In vivo Ca2+ imaging of somatic, dendritic and axonal activity patterns of cortical neurons revealed that both healthy aging and AD-related mutations augment neuronal hyperactivity. The AD-related enhancement occurred even without amyloid deposition and neuroinflammation, mainly due to presenilin-mediated dysfunction of intracellular Ca2+ stores in presynaptic boutons, likely causingmore frequent activation of synaptic NMDA receptors. In mutant but not wild-type mice, store emptying reduced both the frequency and amplitude of presynaptic Ca2+ transients and, most importantly, normalized neuronal network activity. Postsynaptically, the store dysfunction was minor and largely restricted to hyperactive cells. These findings identify presynaptic Ca2+ stores as a key element controlling AD-related neuronal hyperactivity and as a target for disease-modifying treatments. [ABSTRACT FROM AUTHOR]

Aquaporin-4 (AQP4), the principal water channel in astrocytes, is involved in brain water movement, inflammation, and neuroexcitation. In this study, there was strong neuroprotection in mice lacking AQP4 in a model of global cerebral ischemia produced by transient, bilateral carotid artery occlusion (BCAO). Survival and neurological outcome were greatly improved in die AQP4-/- vs. AQP4+/+ mice after occlusion, with large and robust differences in both outbred (CD1) and inbred (C57b1/6) mouse strains without or with mechanical ventilation. Improved survival was also seen in mice lacking the scaffold protein α-syntrophin, which manifest reduced astrocyte water permeability secondary to defective AQP4 plasma membrane targeting. Intracranial pressure elevation and brain water accumulation were much reduced in the AQP4-/-vs. AQP4+/+ mice after carotid artery occlusion, as were blood-brain barrier (BBB) disruption and neuronal loss. Brain slices from AQP4-/- mice showed significantly reduced cell swelling and cyto-toxicity in response to oxygen-glucose deprivation, compared with slices from AQP4+/+ mice. Our findings suggest that die neuroprotective effect of AQP4 deletion in global cerebral ischemia involves reduced astrocyte swelling and brain water accumulation, resulting in reduced BBB disruption, inflammation, and neuron death. AQP4 water transport inhibition may improve survival and neurological outcome after cardiac arrest and in other conditions associated with global cerebral ischemia. [ABSTRACT FROM AUTHOR]

Liver X receptors (LXRs) play an important role in the regulation of cholesterol by regulating several transporters. In this study, we investigated the role of LXRs in the regulation of human organic anion transporter 1 (hOAT1), a major transporter localized in the basolateral membrane of the renal proximal tubule. Exposure of renal S2 cells expressing hOAT1 to LXR agonists (TO901317 and GW3965) and their endogenous ligand [22(R)-hydroxycholesterol] led to the inhibition of hOAT1-mediated [14C]PAH uptake. This inhibition was abolished by coincubation of the above agonists with 22(S)-hydroxycholesterol, an LXR antagonist. Moreover, it was found that the effect of LXR agonists was not mediated by changes in intracellular cholesterol levels. Interestingly, the inhibitory effect of LXRs was enhanced in the presence of 9-cis retinoic acid, a retinoic X receptor agonist. Kinetic analysis revealed that LXR activation decreased the maximum rate of PAH transport (Jmax) but had no effect on the affinity of the transporter (Kt). This result correlated well with data from Western blot analysis, which showed the decrease in hOAT1 expression following LXR activation. Similarly, TO901317 inhibited [14C]PAH uptake by the renal cortical slices as well as decreasing mOAT1 protein expression in mouse kidney. Our findings indicated for the first time that hOAT1 was downregulated by LXR activation in the renal proximal tubule. [ABSTRACT FROM AUTHOR]