RESUMO
Lenadogene nolparvovec (GS010) was developed to treat a point mutation in mitochondrial ND4 that causes Leber hereditary optic neuropathy. GS010 delivers human cDNA encoding wild-type ND4 packaged into an rAAV2/2 vector that transduces retinal ganglion cells, to induce allotopic expression of hybrid mitochondrial ND4. GS010 clinical trials improved best-corrected visual acuity (BCVA) up to 5 years after treatment. Interestingly, unilateral treatment improved BCVA bilaterally. Subsequent studies revealed GS010 DNA in visual tissues contralateral to the injected eye, suggesting migration. Here we tested whether unilateral intraocular pressure (IOP) elevation could influence the transfer of viral ND4 RNA in contralateral tissues after GS010 delivery to the IOP-elevated eye and probed a potential mechanism mediating translocation in mice. We found IOP elevation enhanced viral ND4 RNA transcripts in contralateral visual tissues, including retinas. Using conditional transgenic mice, we depleted astrocytic gap junction connexin 43 (Cx43), required for distant redistribution of metabolic resources between astrocytes during stress. After unilateral IOP elevation and GS010 injection, Cx43 knockdown eradicated ND4 RNA transcript detection in contralateral retinal tissues, while transcript was still detectable in optic nerves. Overall, our study indicates long-range migration of GS010 product to contralateral visual tissues is enhanced by Cx43-linked astrocyte networks.
Assuntos
Astrócitos , Conexina 43 , Camundongos , Humanos , Animais , Astrócitos/metabolismo , Conexina 43/genética , Conexina 43/metabolismo , NADH Desidrogenase/genética , NADH Desidrogenase/metabolismo , Dependovirus/genética , Dependovirus/metabolismo , Vetores Genéticos , Terapia Genética , Camundongos Transgênicos , RNA , DNA Mitocondrial/genéticaRESUMO
BACKGROUND: Cell-to-cell communication is vital for tissues to respond, adapt, and thrive in the prevailing milieu. Several mechanisms mediate intercellular signaling, including tunneling nanotubes, gap junctions, and extracellular vesicles (EV). Depending on local and systemic conditions, EVs may contain cargoes that promote survival, neuroprotection, or pathology. Our understanding of pathologic intercellular signaling has been bolstered by disease models using neurons derived from human pluripotent stems cells (hPSC). METHODS: Here, we used hPSC-derived retinal ganglion cells (hRGC) and the mouse visual system to investigate the influence of modulating EV generation on intercellular trafficking and cell survival. We probed the impact of EV modulation on cell survival by decreasing the catabolism of sphingomyelin into ceramide through inhibition of neutral sphingomyelinase (nSMase), using GW4869. We assayed for cell survival in vitro by probing for annexin A5, phosphatidylserine, viable mitochondria, and mitochondrial reactive oxygen species. In vivo, we performed intraocular injections of GW4869 and measured RGC and superior colliculus neuron density and RGC anterograde axon transport. RESULTS: Following twenty-four hours of dosing hRGCs with GW4869, we found that inhibition of nSMase decreased ceramide and enhanced GM1 ganglioside accumulation. This inhibition also reduced the density of small EVs, increased the density of large EVs, and enriched the pro-apoptotic protein, annexin A5. Reducing nSMase activity increased hRGC apoptosis initiation due to enhanced density and uptake of apoptotic particles, as identified by the annexin A5 binding phospholipid, phosphatidylserine. We assayed intercellular trafficking of mitochondria by developing a coculture system of GW4869-treated and naïve hRGCs. In treated cells, inhibition of nSMase reduced the number of viable mitochondria, while driving mitochondrial reactive oxygen species not only in treated, but also in naive hRGCs added in coculture. In mice, 20 days following a single intravitreal injection of GW4869, we found a significant loss of RGCs and their axonal recipient neurons in the superior colliculus. This followed a more dramatic reduction in anterograde RGC axon transport to the colliculus. CONCLUSION: Overall, our data suggest that perturbing the physiologic catabolism of sphingomyelin by inhibiting nSMase reorganizes plasma membrane associated sphingolipids, alters the profile of neuron-generated EVs, and promotes neurodegeneration in vitro and in vivo by shifting the balance of pro-survival versus -degenerative EVs. Video Abstract.
Assuntos
Esfingomielina Fosfodiesterase , Esfingomielinas , Camundongos , Animais , Humanos , Esfingomielina Fosfodiesterase/metabolismo , Anexina A5 , Espécies Reativas de Oxigênio/metabolismo , Fosfatidilserinas , Ceramidas/metabolismo , Células Ganglionares da Retina/metabolismoRESUMO
In the central nervous system, glycogen-derived bioenergetic resources in astrocytes help promote tissue survival in response to focal neuronal stress. However, our understanding of the extent to which these resources are mobilized and utilized during neurodegeneration, especially in nearby regions that are not actively degenerating, remains incomplete. Here we modeled neurodegeneration in glaucoma, the world's leading cause of irreversible blindness, and measured how metabolites mobilize through astrocyte gap junctions composed of connexin 43 (Cx43). We elevated intraocular pressure in one eye and determined how astrocyte-derived metabolites in the contralateral optic projection responded. Remarkably, astrocyte networks expand and redistribute metabolites along distances even 10 mm in length, donating resources from the unstressed to the stressed projection in response to intraocular pressure elevation. While resource donation improves axon function and visual acuity in the directly stressed region, it renders the donating tissue susceptible to bioenergetic, structural, and physiological degradation. Intriguingly, when both projections are stressed in a WT animal, axon function and visual acuity equilibrate between the two projections even when each projection is stressed for a different length of time. This equilibration does not occur when Cx43 is not present. Thus, Cx43-mediated astrocyte metabolic networks serve as an endogenous mechanism used to mitigate bioenergetic stress and distribute the impact of neurodegenerative disease processes. Redistribution ultimately renders the donating optic nerve vulnerable to further metabolic stress, which could explain why local neurodegeneration does not remain confined, but eventually impacts healthy regions of the brain more broadly.
Assuntos
Astrócitos , Glaucoma/metabolismo , Doenças Neurodegenerativas/metabolismo , Animais , Astrócitos/metabolismo , Astrócitos/fisiologia , Conexina 43/genética , Conexina 43/metabolismo , Feminino , Junções Comunicantes/metabolismo , Pressão Intraocular/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
The structural and biomechanical properties of collagen-rich ocular tissues, such as the sclera, are integral to ocular function. The degradation of collagen in such tissues is associated with debilitating ophthalmic diseases such as glaucoma and myopia, which often lead to visual impairment. Collagen mimetic peptides (CMPs) have emerged as an effective treatment to repair damaged collagen in tissues of the optic projection, such as the retina and optic nerve. In this study, we used atomic force microscopy (AFM) to assess the potential of CMPs in restoring tissue stiffness in the optic nerve head (ONH), including the peripapillary sclera (PPS) and the glial lamina. Using rat ONH tissue sections, we induced collagen damage with MMP-1, followed by treatment with CMP-3 or vehicle. MMP-1 significantly reduced the Young's modulus of both the PPS and the glial lamina, indicating tissue softening. Subsequent CMP-3 treatment partially restored tissue stiffness in both the PPS and the glial lamina. Immunohistochemical analyses revealed reduced collagen fragmentation after MMP-1 digestion in CMP-3-treated tissues compared to vehicle controls. In summary, these results demonstrate the potential of CMPs to restore collagen stiffness and structure in ONH tissues following enzymatic damage. CMPs may offer a promising therapeutic avenue for preserving vision in ocular disorders involving collagen remodeling and degradation.
Assuntos
Disco Óptico , Animais , Disco Óptico/metabolismo , Esclera/metabolismo , Roedores/metabolismo , Metaloproteinase 1 da Matriz/metabolismo , Colágeno/metabolismo , Pressão Intraocular , Fenômenos BiomecânicosRESUMO
BACKGROUND: Leber Hereditary Optic Neuropathy (LHON) is a rare, maternally-inherited mitochondrial disease that primarily affects retinal ganglion cells (RGCs) and their axons in the optic nerve, leading to irreversible, bilateral severe vision loss. Lenadogene nolparvovec gene therapy was developed as a treatment for patients with vision loss from LHON caused by the most prevalent m.11778G > A mitochondrial DNA point mutation in the MT-ND4 gene. Lenadogene nolparvovec is a replication-defective recombinant adeno-associated virus vector 2 serotype 2 (AAV2/2), encoding the human wild-type MT-ND4 protein. Lenadogene nolparvovec was administered by intravitreal injection (IVT) in LHON patients harboring the m.11778G > A ND4 mutation in a clinical development program including one phase 1/2 study (REVEAL), three phase 3 pivotal studies (REVERSE, RESCUE, REFLECT), and one long-term follow-up study (RESTORE, the follow-up of REVERSE and RESCUE patients). CASE PRESENTATION: A 67-year-old woman with MT-ND4 LHON, included in the REVERSE clinical study, received a unilateral IVT of lenadogene nolparvovec in the right eye and a sham injection in the left eye in May 2016, 11.4 months and 8.8 months after vision loss in her right and left eyes, respectively. The patient had a normal brain magnetic resonance imaging with contrast at the time of diagnosis of LHON. Two years after treatment administration, BCVA had improved in both eyes. The product was well tolerated with mild and resolutive anterior chamber inflammation in the treated eye. In May 2019, the patient was diagnosed with a right temporal lobe glioblastoma, IDH-wildtype, World Health Organization grade 4, based on histological analysis of a tumor excision. The brain tumor was assessed for the presence of vector DNA by using a sensitive validated qPCR assay targeting the ND4 sequence of the vector. CONCLUSION: ND4 DNA was not detected (below 15.625 copies/µg of genomic DNA) in DNA extracted from the brain tumor, while a housekeeping gene DNA was detected at high levels. Taken together, this data shows the absence of detection of lenadogene nolparvovec in a brain tumor (glioblastoma) of a treated patient in the REVERSE clinical trial 3 years after gene therapy administration, supporting the long-term favorable safety of lenadogene nolparvovec.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Atrofia Óptica Hereditária de Leber , Idoso , Biópsia , Ensaios Clínicos Fase III como Assunto , Dependovirus , Feminino , Seguimentos , Humanos , Atrofia Óptica Hereditária de Leber/genética , Atrofia Óptica Hereditária de Leber/terapiaRESUMO
Vision loss through the degeneration of retinal ganglion cell (RGC) axons occurs in both chronic and acute conditions that target the optic nerve. These include glaucoma, in which sensitivity to intraocular pressure (IOP) causes early RGC axonal dysfunction, and optic nerve trauma, which causes rapid axon degeneration from the site of injury. In each case, degeneration is irreversible, necessitating new therapeutics that protect, repair, and regenerate RGC axons. Recently, we demonstrated the reparative capacity of using collagen mimetic peptides (CMPs) to heal fragmented collagen in the neuronal extracellular milieu. This was an important step in the development of neuronal-based therapies since neurodegeneration involves matrix metalloproteinase (MMP)-mediated remodeling of the collagen-rich environment in which neurons and their axons exist. We found that intraocular delivery of a CMP comprising single-strand fractions of triple helix human type I collagen prevented early RGC axon dysfunction in an inducible glaucoma model. Additionally, CMPs also promoted neurite outgrowth from dorsal root ganglia, challenged in vitro by partial digestion of collagen. Here, we compared the ability of a CMP sequence to protect RGC axons in both inducible glaucoma and optic nerve crush. A three-week +40% elevation in IOP caused a 67% degradation in anterograde transport to the superior colliculus, the primary retinal projection target in rodents. We found that a single intravitreal injection of CMP during the period of IOP elevation significantly reduced this degradation. The same CMP delivered shortly after optic nerve crush promoted significant axonal recovery during the two-week period following injury. Together, these findings support a novel protective and reparative role for the use of CMPs in both chronic and acute conditions affecting the survival of RGC axons in the optic projection to the brain.
Assuntos
Glaucoma , Células Ganglionares da Retina , Animais , Axônios/metabolismo , Colágeno/metabolismo , Modelos Animais de Doenças , Glaucoma/metabolismo , Pressão Intraocular , Peptídeos/metabolismo , Células Ganglionares da Retina/metabolismoRESUMO
Epithelial cells of multiple types produce and interact with the extracellular matrix to maintain structural integrity and promote healthy function within diverse endogenous tissues. Collagen is a critical component of the matrix, and challenges to collagen's stability in aging, disease, and injury influence survival of adherent epithelial cells. The retinal pigment epithelium (RPE) is important for maintaining proper function of the light-sensitive photoreceptors in the neural retina, in part through synergy with the collagen-rich Bruch's membrane that promotes RPE adherence. Degradation of Bruch's is associated with RPE degeneration, which is implicated early in age-related macular degeneration, a leading cause of irreversible vision loss worldwide. Collagen mimetic peptides (CMPs) effectively repair damage to collagen helices, which are present in all collagens. Our previous work indicates that in doing so, CMPs promote survival and integrity of affected cells and tissues in models of ocular injury and disease, including wounding of corneal epithelial cells. Here, we show that CMPs increase adherence and migration of the ARPE-19 line of human RPE cells challenged by digestion of their collagen substrate. Application of CMPs also reduced both ARPE-19 secretion of pro-inflammatory cytokines (interleukins 6 and 8) and production of reactive oxygen species. Taken together, these results suggest that repairing collagen damaged by aging or other pathogenic processes in the posterior eye could improve RPE adherence and survival and, in doing so, reduce the inflammatory and oxidative stress that perpetuates the cycle of destruction at the root of age-related diseases of the outer retina.
Assuntos
Lâmina Basilar da Corioide , Epitélio Pigmentado da Retina , Colágeno/metabolismo , Colágeno/farmacologia , Humanos , Estresse Oxidativo , Peptídeos/metabolismo , Peptídeos/farmacologia , Epitélio Pigmentado da Retina/metabolismoRESUMO
The nitric oxide-guanylyl cyclase-1-cyclic guanylate monophosphate (NO-GC-1-cGMP) pathway is integral to the control of vascular tone and morphology. Mice lacking the alpha catalytic domain of guanylate cyclase (GC1-/-) develop retinal ganglion cell (RGC) degeneration with age, with only modest fluctuations in intraocular pressure (IOP). Increasing the bioavailability of cGMP in GC1-/- mice prevents neurodegeneration independently of IOP, suggesting alternative mechanisms of retinal neurodegeneration. In continuation to these studies, we explored the hypothesis that dysfunctional cGMP signaling leads to changes in the neurovascular unit that may contribute to RGC degeneration. We assessed retinal vasculature and astrocyte morphology in young and aged GC1-/- and wild type mice. GC1-/- mice exhibit increased peripheral retinal vessel dilation and shorter retinal vessel branching with increasing age compared to Wt mice. Astrocyte cell morphology is aberrant, and glial fibrillary acidic protein (GFAP) density is increased in young and aged GC1-/- mice, with areas of dense astrocyte matting around blood vessels. Our results suggest that proper cGMP signaling is essential to retinal vessel morphology with increasing age. Vascular changed are preceded by alterations in astrocyte morphology which may together contribute to retinal neurodegeneration and loss of visual acuity observed in GC1-/- mice.
Assuntos
Astrócitos , Óxido Nítrico , Animais , Astrócitos/metabolismo , GMP Cíclico/metabolismo , Camundongos , Camundongos Knockout , Óxido Nítrico/metabolismo , Transdução de SinaisRESUMO
Diseases of the brain involve early axon dysfunction that often precedes outright degeneration. Pruning of dendrites and their synapses represents a potential driver of axonopathy by reducing activity. Optic nerve degeneration in glaucoma, the world's leading cause of irreversible blindness, involves early stress to retinal ganglion cell (RGC) axons from sensitivity to intraocular pressure (IOP). This sensitivity also influences survival of RGC dendrites and excitatory synapses in the retina. Here we tested in individual RGCs identified by type the relationship between dendritic organization and axon signaling to light following modest, short-term elevations in pressure. We found dendritic pruning occurred early, by 2 wk of elevation, and independent of whether the RGC responded to light onset (ON cells) or offset (OFF cells). Pruning was similarly independent of ON and OFF in the DBA/2J mouse, a chronic glaucoma model. Paradoxically, all RGCs, even those with significant pruning, demonstrated a transient increase in axon firing in response to the preferred light stimulus that occurred on a backdrop of generally enhanced excitability. The increased response was not through conventional presynaptic signaling, but rather depended on voltage-sensitive sodium channels that increased transiently in the axon. Pruning, axon dysfunction, and deficits in visual acuity did not progress between 2 and 4 wk of elevation. These results suggest neurodegeneration in glaucoma involves an early axogenic response that counters IOP-related stress to excitatory dendritic architecture to slow progression and maintain signaling to the brain. Thus, short-term exposure to elevated IOP may precondition the neural system to further insult.
Assuntos
Axônios/fisiologia , Glaucoma/fisiopatologia , Células Ganglionares da Retina/fisiologia , Animais , Dendritos/fisiologia , Progressão da Doença , Glaucoma/patologia , Humanos , Pressão Intraocular , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Degeneração Neural , Nervo Óptico/fisiopatologia , Células Ganglionares da Retina/patologiaRESUMO
Glaucoma is a multifactorial disease that is conventionally managed with treatments to lower intraocular pressure (IOP). Despite these efforts, many patients continue to lose their vision. The degeneration of retinal ganglion cells (RGCs) and their axons in the optic tract that characterizes glaucoma is similar to neurodegeneration in other age-related disorders of the central nervous system (CNS). Identifying the different molecular signaling pathways that contribute to early neuronal dysfunction can be utilized for neuroprotective strategies that prevent degeneration. The discovery of insulin and its receptor in the CNS and retina led to exploration of the role of insulin signaling in the CNS. Historically, insulin was considered a peripherally secreted hormone that regulated glucose homeostasis, with no obvious roles in the CNS. However, a growing number of pre-clinical and clinical studies have demonstrated the potential of modulating insulin signaling in the treatment of neurodegenerative diseases. This review will highlight the role that insulin signaling plays in RGC neurodegeneration. We will focus on how this pathway can be therapeutically targeted to promote RGC axon survival and preserve vision.
Assuntos
Glaucoma/metabolismo , Insulina/metabolismo , Doenças Neurodegenerativas/metabolismo , Animais , Modelos Animais de Doenças , Glaucoma/patologia , Glaucoma/terapia , Humanos , Terapia de Alvo Molecular , Doenças Neurodegenerativas/patologia , Doenças Neurodegenerativas/terapia , Neuroproteção , Retina/metabolismo , Retina/patologia , Células Ganglionares da Retina/metabolismo , Células Ganglionares da Retina/patologia , Transdução de SinaisRESUMO
Glaucoma is an age-related neurodegenerative disease that is commonly associated with sensitivity to intraocular pressure. The disease selectively targets retinal ganglion cells (RGCs) and constituent axons. RGC axons are rich in voltage-gated sodium channels, which are essential for action potential initiation and regeneration. Here, we identified voltage-dependent sodium channel, NaV1.2, in the retina, examined how this channel contributes to RGC light responses, and monitored NaV1.2 mRNA and protein expression in the retina during progression of modeled glaucoma. We found NaV1.2 is predominately localized in ganglion cell intraretinal axons with dispersed expression in the outer and inner plexiform layers. We showed Phrixotoxin-3, a potent NaV1.2 channel blocker, significantly decreased RGC electrical activity in a dose-dependent manner with an IC50 of 40 nM. Finally, we found four weeks of raised intraocular pressure (30% above baseline) significantly increased NaV1.2 mRNA expression but reduced NaV1.2 protein level in the retina up to 57% (p < 0.001). Following prolonged intraocular pressure elevation, NaV1.2 protein expression particularly diminished at distal sections of ganglion cell intraretinal axons (p ≤ 0.01). Our results suggest NaV1.2 might be a therapeutic target during disease progression to maintain RGC excitability, preserving presynaptic connections through action potential backpropagation.
Assuntos
Axônios/metabolismo , Pressão Intraocular/fisiologia , Canal de Sódio Disparado por Voltagem NAV1.2/metabolismo , Hipertensão Ocular/metabolismo , Células Ganglionares da Retina/metabolismo , Animais , Regulação da Expressão Gênica/fisiologia , Masculino , Camundongos Endogâmicos C57BL , Canal de Sódio Disparado por Voltagem NAV1.2/genética , RNA Mensageiro/genética , Tonometria OcularRESUMO
This review arose from a discussion of regenerative therapies to treat optic nerve degeneration in glaucoma at the 2015 Lasker/IRRF Initiative on Astrocytes and Glaucomatous Neurodegeneration. In addition to the authors, participants included Jonathan Crowston, Andrew Huberman, Elaine Johnson, Richard Lu, Hemai Phatnami, Rebecca Sappington, and Don Zack. Glaucoma is a neurodegenerative disease of the optic nerve, and is the leading cause of irreversible blindness worldwide. The disease progresses as sensitivity to intraocular pressure (IOP) is conveyed through the optic nerve head to distal retinal ganglion cell (RGC) projections. Because the nerve and retina are components of the central nervous system (CNS), their intrinsic regenerative capacity is limited. However, recent research in regenerative therapies has resulted in multiple breakthroughs that may unlock the optic nerve's regenerative potential. Increasing levels of Schwann-cell derived trophic factors and reducing potent cell-intrinsic suppressors of regeneration have resulted in axonal regeneration even beyond the optic chiasm. Despite this success, many challenges remain. RGC axons must be able to form new connections with their appropriate targets in central brain regions and these connections must be retinotopically correct. Furthermore, for new axons penetrating the optic projection, oligodendrocyte glia must provide myelination. Additionally, reactive gliosis and inflammation that increase the regenerative capacity must be outweigh pro-apoptotic processes to create an environment within which maximal regeneration can occur.
Assuntos
Glaucoma/terapia , Degeneração Neural/terapia , Regeneração Nervosa/fisiologia , Neuroglia/fisiologia , Disco Óptico/fisiologia , Doenças do Nervo Óptico/terapia , Medicina Regenerativa , Animais , Humanos , Células Ganglionares da Retina/fisiologiaRESUMO
Glaucoma, a common cause of blindness, is currently treated by intraocular pressure (IOP)-lowering interventions. However, this approach is insufficient to completely prevent vision loss. Here, we evaluate an IOP-independent gene therapy strategy using a modified erythropoietin, EPO-R76E, which has reduced erythropoietic function. We used two models of glaucoma, the murine microbead occlusion model and the DBA/2J mouse. Systemic recombinant adeno-associated virus-mediated gene delivery of EpoR76E (rAAV.EpoR76E) was performed concurrent with elevation of IOP. Axon structure and active anterograde transport were preserved in both models. Vision, as determined by the flash visual evoked potential, was preserved in the DBA/2J. These results show that systemic EpoR76E gene therapy protects retinal ganglion cells from glaucomatous degeneration in two different models. This suggests that EPO targets a component of the neurodegenerative pathway that is common to both models. The efficacy of rAAV.EpoR76E delivered at onset of IOP elevation supports clinical relevance of this treatment.
Assuntos
Axônios/patologia , Eritropoetina/genética , Glaucoma/terapia , Mutação , Nervo Óptico/patologia , Animais , Dependovirus/genética , Modelos Animais de Doenças , Terapia Genética , Vetores Genéticos/administração & dosagem , Glaucoma/genética , Glaucoma/patologia , Humanos , Pressão Intraocular , CamundongosRESUMO
Glaucoma challenges the survival of retinal ganglion cell axons in the optic nerve through processes dependent on both aging and ocular pressure. Relevant stressors likely include complex interplay between axons and astrocytes, both in the retina and optic nerve. In the DBA/2J mouse model of pigmentary glaucoma, early progression involves axonopathy characterized by loss of functional transport prior to outright degeneration. Here we describe novel features of early pathogenesis in the DBA/2J nerve. With age the cross-sectional area of the nerve increases; this is associated generally with diminished axon packing density and survival and increased glial coverage of the nerve. However, for nerves with the highest axon density, as the nerve expands mean cross-sectional axon area enlarges as well. This early expansion was marked by disorganized axoplasm and accumulation of hyperphosphorylated neurofilamants indicative of axonopathy. Axon expansion occurs without loss up to a critical threshold for size (about 0.45-0.50 µm(2)), above which additional expansion tightly correlates with frank loss of axons. As well, early axon expansion prior to degeneration is concurrent with decreased astrocyte ramification with redistribution of processes towards the nerve edge. As axons expand beyond the critical threshold for loss, glial area resumes an even distribution from the center to edge of the nerve. We also found that early axon expansion is accompanied by reduced numbers of mitochondria per unit area in the nerve. Finally, our data indicate that both IOP and nerve expansion are associated with axon enlargement and reduced axon density for aged nerves. Collectively, our data support the hypothesis that diminished bioenergetic resources in conjunction with early nerve and glial remodeling could be a primary inducer of progression of axon pathology in glaucoma.
Assuntos
Astrócitos/patologia , Glaucoma de Ângulo Aberto/patologia , Degeneração Neural/patologia , Doenças do Nervo Óptico/patologia , Nervo Óptico/patologia , Células Ganglionares da Retina/patologia , Animais , Axônios/patologia , Modelos Animais de Doenças , Imageamento Tridimensional , Camundongos , Camundongos Endogâmicos DBA , Degeneração Neural/etiologia , Doenças do Nervo Óptico/etiologia , Fotomicrografia , Fatores de TempoRESUMO
Within the white matter, axonal loss by neurodegeneration is coupled to glial cell changes in gene expression, structure and function commonly termed gliosis. Recently, we described the highly variable expansion of gliosis alebosco@neuro.utah.edu in degenerative optic nerves from the DBA/2J mouse model of chronic, age-related glaucoma. Here, to estimate and compare the levels of axonal loss with the expansion of glial coverage and axonal degeneration in DBA/2J nerves, we combined semiautomatic axon counts with threshold-based segmentation of total glial/scar areas and degenerative axonal profiles in plastic cross-sections. In nerves ranging from mild to severe degeneration, we found that the progression of axonal dropout is coupled to an increase of gliotic area. We detected a strong correlation between axon loss and the aggregate coverage by glial cells and scar, whereas axon loss did not correlate with the small fraction of degenerating profiles. Nerves with low to medium levels of axon loss displayed moderate glial reactivity, consisting of hypertrophic astrocytes, activated microglia and normal distribution of oligodendrocytes, with minimal reorganization of the tissue architecture. In contrast, nerves with extensive axonal loss showed prevalent rearrangement of the nerve, with loss of axon fascicle territories and enlarged or almost continuous gliotic and scar domains, containing reactive astrocytes, oligodendrocytes and activated microglia. These findings support the value of optic nerve gliotic expansion as a quantitative estimate of optic neuropathy that correlates with axon loss, applicable to grade the severity of optic nerve damage in mouse chronic glaucoma.
Assuntos
Glaucoma/patologia , Gliose/complicações , Neuroglia/patologia , Doenças do Nervo Óptico/patologia , Nervo Óptico/patologia , Células Ganglionares da Retina/patologia , Animais , Astrócitos/patologia , Axônios/patologia , Doença Crônica , Modelos Animais de Doenças , Feminino , Glaucoma/complicações , Gliose/patologia , Masculino , Camundongos , Camundongos Endogâmicos DBA , Microscopia Confocal , Doenças do Nervo Óptico/etiologia , FotomicrografiaRESUMO
How neurons respond to stress in degenerative disease is of fundamental importance for identifying mechanisms of progression and new therapeutic targets. Members of the transient receptor potential (TRP) family of cation-selective ion channels are candidates for mediating stress signals, since different subunits transduce a variety of stimuli relevant in both normal and pathogenic physiology. We addressed this possibility for the TRP vanilloid-1 (TRPV1) subunit by comparing how the optic projection of Trpv1(-/-) mice and age-matched C57 controls responds to stress from elevated ocular pressure, the critical stressor in the most common optic neuropathy, glaucoma. Over a 5 week period of elevated pressure induced by microbead occlusion of ocular fluid, Trpv1(-/-) accelerated both degradation of axonal transport from retinal ganglion cells to the superior colliculus and degeneration of the axons themselves in the optic nerve. Ganglion cell body loss, which is normally later in progression, occurred in nasal sectors of Trpv1(-/-) but not C57 retina. Pharmacological antagonism of TRPV1 in rats similarly accelerated ganglion cell axonopathy. Elevated ocular pressure resulted in differences in spontaneous firing rate and action potential threshold current in Trpv1(-/-) ganglion cells compared with C57. In the absence of elevated pressure, ganglion cells in the two strains had similar firing patterns. Based on these data, we propose that TRPV1 may help neurons respond to disease-relevant stressors by enhancing activity necessary for axonal signaling.
Assuntos
Degeneração Neural , Doenças do Nervo Óptico , Células Ganglionares da Retina/patologia , Canais de Cátion TRPV/deficiência , Vias Visuais/patologia , Animais , Axônios/patologia , Toxina da Cólera , Modelos Animais de Doenças , Lateralidade Funcional , Pressão Intraocular/genética , Masculino , Potenciais da Membrana/genética , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Degeneração Neural/etiologia , Degeneração Neural/genética , Degeneração Neural/patologia , Hipertensão Ocular/complicações , Doenças do Nervo Óptico/etiologia , Doenças do Nervo Óptico/genética , Doenças do Nervo Óptico/patologia , Técnicas de Patch-Clamp , Ratos , Células Ganglionares da Retina/metabolismo , Colículos Superiores/metabolismo , Colículos Superiores/patologia , Canais de Cátion TRPV/genéticaRESUMO
Progression of neurodegeneration in disease and injury is influenced by the response of individual neurons to stressful stimuli and whether this response includes mechanisms to counter declining function. Transient receptor potential (TRP) cation channels transduce a variety of disease-relevant stimuli and can mediate diverse stress-dependent changes in physiology, both presynaptic and postsynaptic. Recently, we demonstrated that knock-out or pharmacological inhibition of the TRP vanilloid-1 (TRPV1) capsaicin-sensitive subunit accelerates degeneration of retinal ganglion cell neurons and their axons with elevated ocular pressure, the critical stressor in the most common optic neuropathy, glaucoma. Here we probed the mechanism of the influence of TRPV1 on ganglion cell survival in mouse models of glaucoma. We found that induced elevations of ocular pressure increased TRPV1 in ganglion cells and its colocalization at excitatory synapses to their dendrites, whereas chronic elevation progressively increased ganglion cell Trpv1 mRNA. Enhanced TRPV1 expression in ganglion cells was transient and supported a reversal of the effect of TRPV1 on ganglion cells from hyperpolarizing to depolarizing, which was also transient. Short-term enhancement of TRPV1-mediated activity led to a delayed increase in axonal spontaneous excitation that was absent in ganglion cells from Trpv1(-/-) retina. In isolated ganglion cells, pharmacologically activated TRPV1 mobilized to discrete nodes along ganglion cell dendrites that corresponded to sites of elevated Ca(2+). These results suggest that TRPV1 may promote retinal ganglion cell survival through transient enhancement of local excitation and axonal activity in response to ocular stress.
Assuntos
Células Ganglionares da Retina/fisiologia , Estresse Fisiológico/fisiologia , Canais de Cátion TRPV/fisiologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Cálcio/metabolismo , Capsaicina/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Modelos Animais de Doenças , Diterpenos/farmacologia , Dopamina/análogos & derivados , Dopamina/farmacologia , Glaucoma/metabolismo , Glaucoma/fisiopatologia , Pressão Intraocular/fisiologia , Camundongos , Camundongos Knockout , Cultura Primária de Células , Células Ganglionares da Retina/efeitos dos fármacos , Células Ganglionares da Retina/metabolismo , Canais de Cátion TRPV/agonistas , Canais de Cátion TRPV/antagonistas & inibidores , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismoRESUMO
As in other age-related neurodegenerative diseases, progression of neurodegeneration in glaucoma involves early axonopathy. In glaucoma, this is marked by degradation of active transport along retinal ganglion cell (RGC) axons projecting from the retina to the brain. In experimental systems, transport degradation occurs first in the most distal site in the RGC projection, the superior colliculus (SC) of the midbrain. Even as degradation progresses from one retinotopic sector to the next, important structures in the affected sectors persist, including synapses from RGC axon terminals onto SC neurons. This structural persistence is accompanied by focally increased brain-derived neurotrophic factor in hypertrophic SC astrocyte glia and defines a therapeutic window of opportunity. Thus, central brain structures in glaucoma may respond to disease-relevant stress by induction of mechanisms useful for maintaining retinal signals.
Assuntos
Glaucoma/patologia , Glaucoma/fisiopatologia , Neurônios/fisiologia , Recuperação de Função Fisiológica/fisiologia , Vias Visuais/fisiopatologia , HumanosRESUMO
Astrocytes provide metabolic, structural, and synaptic support to neurons in normal physiology and also contribute widely to pathogenic processes in response to stress or injury. Reactive astrocytes can undergo cytoskeletal reorganization and increase migration through changes in intracellular Ca(2+) mediated by a variety of potential modulators. Here we tested whether migration of isolated retinal astrocytes following mechanical injury (scratch wound) involves the transient receptor potential vanilloid-1 channel (TRPV1), which contributes to Ca(2+)-mediated cytoskeletal rearrangement and migration in other systems. Application of the TRPV1-specific antagonists, capsazepine (CPZ) or 5'-iodoresiniferatoxin (IRTX), slowed migration by as much as 44%, depending on concentration. In contrast, treatment with the TRPV1-specific agonists, capsaicin (CAP) or resiniferatoxin (RTX) produced only a slight acceleration over a range of concentrations. Chelation of extracellular Ca(2+) with EGTA (1 mM) slowed astrocyte migration by 35%. Ratiometric imaging indicated that scratch wound induced a sharp 20% rise in astrocyte Ca(2+) that dissipated with distance from the wound. Treatment with IRTX both slowed and dramatically reduced the scratch-induced Ca(2+) increase. Both CPZ and IRTX influenced astrocyte cytoskeletal organization, especially near the wound edge. Taken together, our results indicate that astrocyte mobilization in response to mechanical stress involves influx of extracellular Ca(2+) and cytoskeletal changes in part mediated by TRPV1 activation.
Assuntos
Astrócitos/fisiologia , Movimento Celular/fisiologia , Estresse Mecânico , Canais de Cátion TRPV/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Cálcio/metabolismo , Técnicas de Cultura de Células , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/fisiologia , Espaço Extracelular/metabolismo , Espaço Intracelular/metabolismo , Camundongos Endogâmicos C57BL , Ratos Sprague-Dawley , Retina/lesões , Retina/fisiopatologia , Canais de Cátion TRPV/agonistas , Canais de Cátion TRPV/antagonistas & inibidoresRESUMO
Astrocytes serve multiple roles in helping to maintain homeostatic physiology of central nervous system tissue, ranging from metabolic support to coupling between vascular and neural elements. Astrocytes are especially critical in axonal tracts such as the optic nerve, where axons propagate energy-demanding action potentials great distances. In disease, astrocyte remodeling is a dynamic, multifaceted process that is often over-simplified between states of quiescence and reactivity. In glaucoma, axon degeneration in the optic nerve is characterized by progressive stages. So too is astrocyte remodeling. Here, using quantitative analysis of light and electron micrographs of myelinated optic nerve sections from the DBA/2J mouse model of glaucoma, we offer further insight into how astrocyte organization reflects stages of degeneration. This analysis indicates that even as axons degenerate, astrocyte gliosis in the nerve increases without abject proliferation, similar to results in the DBA/2J retina. Gliosis is accompanied by reorganization. As axons expand prior to frank degeneration, astrocyte processes retract from the extra-axonal space and reorient towards the nerve edge. After a critical threshold of expansion, axons drop out, and astrocyte processes distribute more evenly across the nerve reflecting gliosis. This multi-stage process likely reflects local rather than global cues from axons and the surrounding tissue that induce rapid reorganization to promote axon survival and extend functionality of the nerve.