Influence of retinal NMDA receptor activity during autoimmune optic neuritis.

Autoimmune optic neuritis (AON), a model of multiple sclerosis-associated optic neuritis, is accompanied by degeneration of retinal ganglion cells (RGCs) and optic nerve demyelination and axonal loss. In order to investigate the role of N-methyl-d-aspartate (NMDA) receptors in mediating RGC degeneration, upstream changes in the optic nerve actin cytoskeleton and associated deterioration in visual function, we induced AON in Brown Norway rats by immunization with myelin oligodendrocyte glycoprotein. Subsequently, visual acuity was assessed by recording visual evoked potentials and electroretinograms prior to extraction of optic nerves for western blot analysis and retinas for quantification of RGCs. As previously reported, in Brown Norway rats RGC degeneration is observed prior to onset of immune cell infiltration and demyelination of the optic nerves. However, within the optic nerve, destabilization of the actin cytoskeleton could be seen as indicated by an increase in the globular to filamentous actin ratio. Interestingly, these changes could be mimicked by intravitreal injection of glutamate, and similarly blocked by application of the NMDA receptor blocker MK-801, leading us to propose that prior to optic nerve lesion formation, NMDA receptor activation within the retina leads to retinal calcium accumulation, actin destabilization within the optic nerve as well as a deterioration of visual acuity during AON.

Preclinical stress originates in the rat optic nerve head during development of autoimmune optic neuritis.

Optic neuritis is a common manifestation of multiple sclerosis, an inflammatory demyelinating disease of the CNS. Although it is the presenting symptom in many cases, the initial events are currently unknown. However, in the earliest stages of autoimmune optic neuritis in rats, pathological changes are already apparent such as microglial activation and disturbances in myelin ultrastructure of the optic nerves. αB-crystallin is a heat-shock protein induced in cells undergoing cellular stress and has been reported to be up-regulated in both multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis. Therefore, we wished to investigate the timing and localization of its expression in autoimmune optic neuritis. Although loss of oligodendrocytes was not observed until the later disease stages accompanying immune cell infiltration and demyelination, an increase in oligodendrocyte αB-crystallin was observed during the preclinical stages. This was most pronounced within the optic nerve head and was associated with areas of IgG deposition. Since treatment of isolated oligodendrocytes with sera from myelin oligodendrocyte glycoprotein (MOG)-immunized animals induced an increase in αB-crystallin expression, as did passive transfer of sera from MOG-immunized animals to unimmunized recipients, we propose that the partially permeable blood-brain barrier of the optic nerve head may present an opportunity for blood-borne components such as anti-MOG antibodies to come into contact with oligodendrocytes as one of the earliest events in disease development.

Preclinical retinal neurodegeneration in a model of multiple sclerosis.

Neurodegeneration plays a major role in multiple sclerosis (MS), in which it is thought to be the main determinant of permanent disability. However, the relationship between the immune response and the onset of neurodegeneration is still a matter of debate. Moreover, recent findings in MS patients raised the question of whether primary neurodegenerative changes can occur in the retina independent of optic nerve inflammation. Using a rat model of MS that frequently leads to optic neuritis, we have investigated the interconnection between neurodegenerative and inflammatory changes in the retina and the optic nerves with special focus on preclinical disease stages. We report that, before manifestation of optic neuritis, characterized by inflammatory infiltration and demyelination of the optic nerve, degeneration of retinal ganglion cell bodies had already begun and ultrastructural signs of axon degeneration could be detected. In addition, we observed an early activation of resident microglia in the retina. In the optic nerve, the highest density of activated microglia was found within the optic nerve head. In parallel, localized breakdown in the integrity of the blood-retinal barrier and aberrations in the organization of the blood-brain barrier marker aquaporin-4 in the optic nerves were observed during the preclinical phase, before onset of optic neuritis. From these findings, we conclude that early and subtle inflammatory changes in the retina and/or the optic nerve head reminiscent of those suggested for preclinical MS lesions may initiate the process of neurodegeneration in the retina before major histopathological signs of MS become manifest.

Visualizing Synaptic Multi-Protein Patterns of Neuronal Tissue With DNA-Assisted Single-Molecule Localization Microscopy.

The development of super-resolution microscopy (SRM) has widened our understanding of biomolecular structure and function in biological materials. Imaging multiple targets within a single area would elucidate their spatial localization relative to the cell matrix and neighboring biomolecules, revealing multi-protein macromolecular structures and their functional co-dependencies. SRM methods are, however, limited to the number of suitable fluorophores that can be imaged during a single acquisition as well as the loss of antigens during antibody washing and restaining for organic dye multiplexing. We report the visualization of multiple protein targets within the pre- and postsynapse in 350-400 nm thick neuronal tissue sections using DNA-assisted single-molecule localization microscopy (SMLM). In a single labeling step, antibodies conjugated with short DNA oligonucleotides visualized multiple targets by sequential exchange of fluorophore-labeled complementary oligonucleotides present in the imaging buffer. This approach avoids potential effects on structural integrity when using multiple rounds of immunolabeling and eliminates chromatic aberration, because all targets are imaged using a single excitation laser wavelength. This method proved robust for multi-target imaging in semi-thin tissue sections with a lateral resolution better than 25 nm, paving the way toward structural cell biology with single-molecule SRM.

Early Nodal and Paranodal Disruption in Autoimmune Optic Neuritis.

Disturbances in the nodes of Ranvier are an early phenomenon in many CNS disorders, including the autoimmune demyelinating disease multiple sclerosis (MS). Using an animal model of optic neuritis, a common early symptom of MS, we have investigated nodal and paranodal compartments in the optic nerve during disease progression. Both nodes and paranodes, as identified by immunohistochemistry against sodium channels (Nav) and Caspr, respectively, were observed to increase in length during the late induction phase of the disease, prior to onset of the demyelination and immune cell infiltration characteristic of optic neuritis. These changes were correlated with both axonal stress and microglial/macrophage activation, and were most apparent in the vicinity of the retrobulbar optic nerve head, the unmyelinated region of the optic nerve where retinal ganglion cell axons exit the retina. Using intravitreal glutamate injection as a model of a primary retinal insult, we demonstrate that this can induce similar nodal and paranodal changes. This may suggest that onset of neurodegeneration in the absence of demyelination, as reported in several studies into the nonaffected eyes of MS patients, may give rise to subtle disturbances in the axo-glial junction.

Murine Autoimmune Optic Neuritis Is Not Phenotypically Altered by the Retinal Degeneration 8 Mutation.

Purpose: To investigate whether the presence of the retinal degeneration 8 (rd8) mutation in C57BL/6 mice alters the phenotype of autoimmune optic neuritis (AON). Methods: C57BL/6J and C57BL/6N mice were genotyped for the rd8 mutation and fundus analyses and examination of retinal layer morphology were performed in vivo by scanning laser ophthalmoscopy and optical coherence tomography. Visual function was assessed by recording electroretinographs, and visual evoked potentials and retinae and optic nerves were assessed histopathologically. Retinal ganglion cell numbers were determined by retrograde labeling with fluorogold. Mice were then immunized with myelin oligodendrocyte glycoprotein 35-55 to induce AON before assessment of retinal ganglion cell degeneration, inflammatory infiltration of retinae and optic nerves, and demyelination. Furthermore, visual function was assessed by visual evoked potentials. Results: All C57BL/6N mice were homozygous for the mutation (Crb1rd8/rd8) and had pathology typical of the rd8 mutation; however, this was not seen in the C57BL/6J (Crb1wt/wt) mice. Following induction of AON, no differences were seen between the Crb1rd8/rd8 and Crb1wt/wt mice regarding disease parameters nor regarding inner retinal degeneration either in the retina as a whole or in the inferior nasal quadrant. Conclusions: The presence of the rd8 mutation in C57BL/6 mice does not affect the course of AON and should not provide a confounding factor in the interpretation of experimental results obtained in this model. However, it could be dangerous in other models of ocular pathology.

Tryptophan-2,3-Dioxygenase (TDO) deficiency is associated with subclinical neuroprotection in a mouse model of multiple sclerosis.

The catabolism of tryptophan to immunosuppressive and neuroactive kynurenines is a key metabolic pathway regulating immune responses and neurotoxicity. The rate-limiting step is controlled by indoleamine-2,3-dioxygenase (IDO) and tryptophan-2,3-dioxygenase (TDO). IDO is expressed in antigen presenting cells during immune reactions, hepatic TDO regulates blood homeostasis of tryptophan and neuronal TDO influences neurogenesis. While the role of IDO has been described in multiple immunological settings, little is known about TDO's effects on the immune system. TDO-deficiency is neuroprotective in C. elegans and Drosophila by increasing tryptophan and specific kynurenines. Here we have determined the role of TDO in autoimmunity and neurodegeneration in experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. We created reporter-TDO mice for in vivo imaging to show that hepatic but not CNS TDO expression is activated during EAE. TDO deficiency did not influence myelin-specific T cells, leukocyte infiltration into the CNS, demyelination and disease activity. TDO-deficiency protected from neuronal loss in the spinal cord but not in the optic nerves. While this protection did not translate to an improved overt clinical outcome, our data suggest that spatially distinct neuroprotection is conserved in mammals and support TDO as a potential target for treatment of diseases associated with neurodegeneration.

Antibody-mediated inhibition of TNFR1 attenuates disease in a mouse model of multiple sclerosis.

Tumour necrosis factor (TNF) is a proinflammatory cytokine that is known to regulate inflammation in a number of autoimmune diseases, including multiple sclerosis (MS). Although targeting of TNF in models of MS has been successful, the pathological role of TNF in MS remains unclear due to clinical trials where the non-selective inhibition of TNF resulted in exacerbated disease. Subsequent experiments have indicated that this may have resulted from the divergent effects of the two TNF receptors, TNFR1 and TNFR2. Here we show that the selective targeting of TNFR1 with an antagonistic antibody ameliorates symptoms of the most common animal model of MS, experimental autoimmune encephalomyelitis (EAE), when given following both a prophylactic and therapeutic treatment regime. Our results demonstrate that antagonistic TNFR1-specific antibodies may represent a therapeutic approach for the treatment of MS in the future.