RESUMO
BACKGROUND: The risk of glaucoma increases significantly with age and exposure to elevated intraocular pressure, two factors linked with neuroinflammation. The complement cascade is a complex immune process with many bioactive end-products, including mediators of inflammation. Complement cascade activation has been shown in glaucoma patients and models of glaucoma. However, the function of complement-mediated inflammation in glaucoma is largely untested. Here, the complement peptide C3a receptor 1 was genetically disrupted in DBA/2J mice, an ocular hypertensive model of glaucoma, to test its contribution to neurodegeneration. METHODS: A null allele of C3ar1 was backcrossed into DBA/2J mice. Development of iris disease, ocular hypertension, optic nerve degeneration, retinal ganglion cell activity, loss of RGCs, and myeloid cell infiltration in C3ar1-deficient and sufficient DBA/2J mice were compared across multiple ages. RNA sequencing was performed on microglia from primary culture to determine global effects of C3ar1 on microglia gene expression. RESULTS: Deficiency in C3ar1 lowered the risk of degeneration in ocular hypertensive mice without affecting intraocular pressure elevation at 10.5 months of age. Differences were found in the percentage of mice affected, but not in individual characteristics of disease progression. The protective effect of C3ar1 deficiency was then overcome by additional aging and ocular hypertensive injury. Microglia and other myeloid-derived cells were the primary cells identified that express C3ar1. In the absence of C3ar1, microglial expression of genes associated with neuroinflammation and other immune functions were differentially expressed compared to WT. A network analysis of these data suggested that the IL10 signaling pathway is a major interaction partner of C3AR1 signaling in microglia. CONCLUSIONS: C3AR1 was identified as a damaging neuroinflammatory factor. These data help suggest complement activation causes glaucomatous neurodegeneration through multiple mechanisms, including inflammation. Microglia and infiltrating myeloid cells expressed high levels of C3ar1 and are the primary candidates to mediate its effects. C3AR1 appeared to be a major regulator of microglia reactivity and neuroinflammatory function due to its interaction with IL10 signaling and other immune related pathways. Targeting myeloid-derived cells and C3AR1 signaling with therapies is expected to add to or improve neuroprotective therapeutic strategies.
Assuntos
Degeneração Neural/metabolismo , Nervo Óptico/metabolismo , Receptores de Complemento/biossíntese , Receptores de Complemento/deficiência , Animais , Animais Recém-Nascidos , Células Cultivadas , Feminino , Redes Reguladoras de Genes/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos DBA , Camundongos Knockout , Camundongos Transgênicos , Degeneração Neural/genética , Degeneração Neural/patologia , Nervo Óptico/patologia , Receptores de Complemento/genéticaRESUMO
BACKGROUND: Glaucoma is characterized by the progressive dysfunction and loss of retinal ganglion cells. Recent work in animal models suggests that a critical neuroinflammatory event damages retinal ganglion cell axons in the optic nerve head during ocular hypertensive injury. We previously demonstrated that monocyte-like cells enter the optic nerve head in an ocular hypertensive mouse model of glaucoma (DBA/2 J), but their roles, if any, in mediating axon damage remain unclear. METHODS: To understand the function of these infiltrating monocyte-like cells, we used RNA-sequencing to profile their transcriptomes. Based on their pro-inflammatory molecular signatures, we hypothesized and confirmed that monocyte-platelet interactions occur in glaucomatous tissue. Furthermore, to test monocyte function we used two approaches to inhibit their entry into the optic nerve head: (1) treatment with DS-SILY, a peptidoglycan that acts as a barrier to platelet adhesion to the vessel wall and to monocytes, and (2) genetic targeting of Itgam (CD11b, an immune cell receptor that enables immune cell extravasation). RESULTS: Monocyte specific RNA-sequencing identified novel neuroinflammatory pathways early in glaucoma pathogenesis. Targeting these processes pharmacologically (DS-SILY) or genetically (Itgam / CD11b knockout) reduced monocyte entry and provided neuroprotection in DBA/2 J eyes. CONCLUSIONS: These data demonstrate a key role of monocyte-like cell extravasation in glaucoma and demonstrate that modulating neuroinflammatory processes can significantly lessen optic nerve injury.
Assuntos
Glaucoma/patologia , Monócitos/patologia , Degeneração Neural/patologia , Animais , Quimiotaxia de Leucócito , Camundongos , Nervo Óptico/patologiaRESUMO
BACKGROUND: We previously reported a profound long-term neuroprotection subsequent to a single radiation-therapy in the DBA/2J mouse model of glaucoma. This neuroprotection prevents entry of monocyte-like immune cells into the optic nerve head during glaucoma. Gene expression studies in radiation-treated mice implicated Glycam1 in this protection. Glycam1 encodes a proteoglycan ligand for L-selectin and is an excellent candidate to modulate immune cell entry into the eye. Here, we experimentally test the hypothesis that radiation-induced over-expression of Glycam1 is a key component of the neuroprotection. METHODS: We generated a null allele of Glycam1 on a DBA/2J background. Gene and protein expression of Glycam1, monocyte entry into the optic nerve head, retinal ganglion cell death, and axon loss in the optic nerve were assessed. RESULTS: Radiation therapy potently inhibits monocyte entry into the optic nerve head and prevents retinal ganglion cell death and axon loss. DBA/2J mice carrying a null allele of Glycam1 show increased monocyte entry and increased retinal ganglion cell death and axon loss following radiation therapy, but the majority of optic nerves were still protected by radiation therapy. CONCLUSIONS: Although GlyCAM1 is an L-selectin ligand, its roles in immunity are not yet fully defined. The current study demonstrates a partial role for GlyCAM1 in radiation-mediated protection. Furthermore, our results clearly show that GlyCAM1 levels modulate immune cell entry from the vasculature into neural tissues. As Glycam1 deficiency has a more profound effect on cell entry than on neurodegeneration, further experiments are needed to precisely define the role of monocyte entry in DBA/2J glaucoma. Nevertheless, GlyCAM1's function as a negative regulator of extravasation may lead to novel therapeutic strategies for an array of common conditions involving inflammation.
Assuntos
Glaucoma/metabolismo , Glaucoma/radioterapia , Monócitos/metabolismo , Mucinas/biossíntese , Mucinas/efeitos da radiação , Disco Óptico/metabolismo , Animais , Feminino , Glaucoma/prevenção & controle , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Disco Óptico/irrigação sanguínea , Nervo Óptico/irrigação sanguínea , Nervo Óptico/metabolismoRESUMO
Various immune response pathways are altered during early, predegenerative stages of glaucoma; however, whether the early immune responses occur secondarily to or independently of neuronal dysfunction is unclear. To investigate this relationship, we used the Wlds allele, which protects from axon dysfunction. We demonstrate that DBA/2J.Wlds mice develop high intraocular pressure (IOP) but are protected from retinal ganglion cell (RGC) dysfunction and neuroglial changes that otherwise occur early in DBA/2J glaucoma. Despite this, immune pathways are still altered in DBA/2J.Wlds mice. This suggests that immune changes are not secondary to RGC dysfunction or altered neuroglial interactions, but may be directly induced by the increased strain imposed by high IOP. One early immune response following IOP elevation is up-regulation of complement C3 in astrocytes of DBA/2J and DBA/2J.Wlds mice. Unexpectedly, because the disruption of other complement components, such as C1Q, is protective in glaucoma, C3 deficiency significantly increased the number of DBA/2J eyes with nerve damage and RGC loss at an early time point after IOP elevation. Transcriptional profiling of C3-deficient cultured astrocytes implicated EGFR signaling as a hub in C3-dependent responses. Treatment with AG1478, an EGFR inhibitor, also significantly increased the number of DBA/2J eyes with glaucoma at the same early time point. These findings suggest that C3 protects from early glaucomatous damage, a process that may involve EGFR signaling and other immune responses in the optic nerve head. Therefore, therapies that target specific components of the complement cascade, rather than global inhibition, may be more applicable for treating human glaucoma.
Assuntos
Complemento C3/imunologia , Glaucoma/imunologia , Células Ganglionares da Retina/imunologia , Regulação para Cima/imunologia , Animais , Complemento C3/genética , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Receptores ErbB/imunologia , Glaucoma/genética , Glaucoma/patologia , Glaucoma/prevenção & controle , Pressão Intraocular/imunologia , Camundongos , Camundongos Endogâmicos DBA , Camundongos Knockout , Nervo Óptico/imunologia , Nervo Óptico/patologia , Quinazolinas/farmacologia , Células Ganglionares da Retina/patologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Tirfostinas/farmacologiaRESUMO
Primary open-angle glaucoma (POAG) is characterized by progressive neurodegeneration of retinal ganglion cells (RGCs). Why RGCs degenerate in low-pressure POAG remains poorly understood. To gain mechanistic insights, we developed a novel mouse model based on a mutation in human optineurin associated with hereditary, low-pressure POAG. This mouse improves the design and phenotype of currently available optineurin mice, which showed high global overexpression. Although both 18-month-old optineurin and nontransgenic control mice showed an age-related decrease in healthy axons and RGCs, the expression of mutant optineurin enhanced axonal degeneration and decreased RGC survival. Mouse visual function was determined using visual evoked potentials, which revealed specific visual impairment in contrast sensitivity. The E50K optineurin transgenic mouse described here exhibited clinical features of POAG and may be useful for mechanistic dissection of POAG and therapeutic development.
Assuntos
Proteínas do Olho/genética , Glaucoma de Ângulo Aberto/genética , Mutação , Transtornos da Visão/genética , Animais , Axônios/patologia , Proteínas de Ciclo Celular , Sobrevivência Celular/genética , Modelos Animais de Doenças , Potenciais Evocados Visuais , Glaucoma de Ângulo Aberto/patologia , Humanos , Proteínas de Membrana Transportadoras , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Degeneração Neural/genética , Células Ganglionares da Retina/patologia , Transtornos da Visão/patologia , Transtornos da Visão/fisiopatologiaRESUMO
Controlled, wireless neuromodulation using miniature implantable devices is a long-sought goal in neuroscience. It will allow many studies and treatments that are otherwise impractical. Recent studies demonstrate advances in neuromodulation through optogenetics, but test animals are typically tethered, severely limiting experimental possibilities. Existing nontethered optical stimulators either deliver light through a cranial window limiting applications to superficial layers of the brain, are not widely accessible due to highly specialized fabrication techniques, or do not demonstrate robust and flexible control of the optical power emitted. To overcome these limitations, we have developed a novel, miniature, wireless, deep-brain, modular optical stimulator with controllable stimulation parameters for use in optogenetic experiments. We demonstrate its use in a behavioral experiment targeting a deep brain structure in freely behaving mice. To allow its rapid and widespread adoption, we developed this stimulator using commercially available components. The modular and accessible optogenetic stimulator presented advances the wireless toolset available for freely behaving animal experiments.
Assuntos
Estimulação Encefálica Profunda/instrumentação , Fibras Ópticas , Optogenética/instrumentação , Tecnologia sem Fio/instrumentação , Potenciais de Ação/fisiologia , Animais , Comportamento Animal , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/fisiologia , Humanos , Cristais Líquidos , Camundongos , Camundongos Transgênicos , Estimulação Luminosa , Simportadores/genética , Simportadores/fisiologia , Área Tegmentar Ventral/fisiologiaRESUMO
The endothelin system is implicated in various human and animal glaucomas. Targeting the endothelin system has great promise as a treatment for human glaucoma, but the cell types involved and the exact mechanisms of action are not clearly elucidated. Here, we report a detailed characterization of the endothelin system in specific cell types of the optic nerve head (ONH) during glaucoma in DBA/2J mice. First, we show that key components of the endothelin system are expressed in multiple cell types. We discover that endothelin 2 (EDN2) is expressed in astrocytes as well as microglia/monocytes in the ONH. The endothelin receptor type A (Ednra) is expressed in vascular endothelial cells, while the endothelin receptor type B (Ednrb) receptor is expressed in ONH astrocytes. Second, we show that Macitentan treatment protects from glaucoma. Macitentan is a novel, orally administered, dual endothelin receptor antagonist with greater affinity, efficacy and safety than previous antagonists. Finally, we test the combinatorial effect of targeting both the endothelin and complement systems as a treatment for glaucoma. Similar to endothelin, the complement system is implicated in a variety of human and animal glaucomas, and has great promise as a treatment target. We discovered that combined targeting of the endothelin (Bosentan) and complement (C1qa mutation) systems is profoundly protective. Remarkably, 80% of DBA/2J eyes subjected to this combined inhibition developed no detectable glaucoma. This opens an exciting new avenue for neuroprotection in glaucoma.
Assuntos
Complemento C1q/metabolismo , Endotelina-2/metabolismo , Glaucoma/complicações , Degeneração Neural/etiologia , Degeneração Neural/terapia , Receptor de Endotelina A/metabolismo , Animais , Astrócitos/metabolismo , Bosentana , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Antagonistas do Receptor de Endotelina A/uso terapêutico , Glaucoma/patologia , Humanos , Camundongos , Camundongos Endogâmicos DBA , Degeneração Neural/patologia , Pirimidinas/uso terapêutico , Células Ganglionares da Retina/efeitos dos fármacos , Células Ganglionares da Retina/metabolismo , Células Ganglionares da Retina/patologia , Sulfonamidas/metabolismo , Sulfonamidas/uso terapêuticoRESUMO
Glaucoma is a complex disease affecting an estimated 70 million people worldwide, characterised by the progressive degeneration of retinal ganglion cells and accompanying visual field loss. The common site of damage to retinal ganglion cells is thought to be at the optic nerve head, however evidence from other optic neuropathies and neurodegenerative disorders suggests that dendritic structures undergo a prolonged period of atrophy that may accompany or even precede soma loss and neuronal cell death. Using the DBA/2J mouse model of glaucoma this investigation aims to elucidate the impact of increasing intraocular pressure on retinal ganglion cell dendrites using DBA/2J mice that express YFP throughout the retinal ganglion cells driven by Thy1 (DBA/2J.Thy1(YFP)) and DiOlistically labelled retinal ganglion cells in DBA/2J mice. Here we show retinal ganglion cell dendritic degeneration in DiOlistically labelled DBA/2J retinal ganglion cells but not in the DBA/2J.Thy1(YFP) retinal ganglion cells suggesting that a potential downregulation of Thy1 allows only 'healthy' retinal ganglion cells to express YFP. These data may highlight alternative pathways to retinal ganglion cell loss in DBA/2J glaucoma.