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The intrinsic mechanisms that regulate neurotoxic versus neuroprotective astrocyte phenotypes and their effects on central nervous system degeneration and repair remain poorly understood. Here we show that injured white matter astrocytes differentiate into two distinct C3-positive and C3-negative reactive populations, previously simplified as neurotoxic (A1) and neuroprotective (A2)1,2, which can be further subdivided into unique subpopulations defined by proliferation and differential gene expression signatures. We find the balance of neurotoxic versus neuroprotective astrocytes is regulated by discrete pools of compartmented cyclic adenosine monophosphate derived from soluble adenylyl cyclase and show that proliferating neuroprotective astrocytes inhibit microglial activation and downstream neurotoxic astrocyte differentiation to promote retinal ganglion cell survival. Finally, we report a new, therapeutically tractable viral vector to specifically target optic nerve head astrocytes and show that raising nuclear or depleting cytoplasmic cyclic AMP in reactive astrocytes inhibits deleterious microglial or macrophage cell activation and promotes retinal ganglion cell survival after optic nerve injury. Thus, soluble adenylyl cyclase and compartmented, nuclear- and cytoplasmic-localized cyclic adenosine monophosphate in reactive astrocytes act as a molecular switch for neuroprotective astrocyte reactivity that can be targeted to inhibit microglial activation and neurotoxic astrocyte differentiation to therapeutic effect. These data expand on and define new reactive astrocyte subtypes and represent a step towards the development of gliotherapeutics for the treatment of glaucoma and other optic neuropathies.
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Astrócitos , Neuroproteção , Adenilil Ciclases/metabolismo , Astrócitos/citologia , Astrócitos/enzimologia , Astrócitos/metabolismo , Diferenciação Celular , Núcleo Celular/metabolismo , Sobrevivência Celular , AMP Cíclico/metabolismo , Citoplasma/metabolismo , Macrófagos/metabolismo , Macrófagos/patologia , Microglia/metabolismo , Microglia/patologia , Traumatismos do Nervo Óptico/metabolismo , Traumatismos do Nervo Óptico/patologia , Traumatismos do Nervo Óptico/terapia , Células Ganglionares da Retina/citologia , Células Ganglionares da Retina/metabolismo , Substância Branca/metabolismo , Substância Branca/patologia , Glaucoma/patologia , Glaucoma/terapiaRESUMO
Multiphoton microscopy can resolve fluorescent structures and dynamics deep in scattering tissue and has transformed neural imaging, but applying this technique in vivo can be limited by the mechanical and optical constraints of conventional objectives. Short working distance objectives can collide with compact surgical windows or other instrumentation and preclude imaging. Here we present an ultra-long working distance (20 mm) air objective called the Cousa objective. It is optimized for performance across multiphoton imaging wavelengths, offers a more than 4 mm2 field of view with submicrometer lateral resolution and is compatible with commonly used multiphoton imaging systems. A novel mechanical design, wider than typical microscope objectives, enabled this combination of specifications. We share the full optical prescription, and report performance including in vivo two-photon and three-photon imaging in an array of species and preparations, including nonhuman primates. The Cousa objective can enable a range of experiments in neuroscience and beyond.
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Corantes , Microscopia de Fluorescência por Excitação Multifotônica , Animais , Microscopia de Fluorescência por Excitação Multifotônica/métodosRESUMO
Neurons have recently emerged as essential cellular constituents of the tumour microenvironment, and their activity has been shown to increase the growth of a diverse number of solid tumours1. Although the role of neurons in tumour progression has previously been demonstrated2, the importance of neuronal activity to tumour initiation is less clear-particularly in the setting of cancer predisposition syndromes. Fifteen per cent of individuals with the neurofibromatosis 1 (NF1) cancer predisposition syndrome (in which tumours arise in close association with nerves) develop low-grade neoplasms of the optic pathway (known as optic pathway gliomas (OPGs)) during early childhood3,4, raising the possibility that postnatal light-induced activity of the optic nerve drives tumour initiation. Here we use an authenticated mouse model of OPG driven by mutations in the neurofibromatosis 1 tumour suppressor gene (Nf1)5 to demonstrate that stimulation of optic nerve activity increases optic glioma growth, and that decreasing visual experience via light deprivation prevents tumour formation and maintenance. We show that the initiation of Nf1-driven OPGs (Nf1-OPGs) depends on visual experience during a developmental period in which Nf1-mutant mice are susceptible to tumorigenesis. Germline Nf1 mutation in retinal neurons results in aberrantly increased shedding of neuroligin 3 (NLGN3) within the optic nerve in response to retinal neuronal activity. Moreover, genetic Nlgn3 loss or pharmacological inhibition of NLGN3 shedding blocks the formation and progression of Nf1-OPGs. Collectively, our studies establish an obligate role for neuronal activity in the development of some types of brain tumours, elucidate a therapeutic strategy to reduce OPG incidence or mitigate tumour progression, and underscore the role of Nf1mutation-mediated dysregulation of neuronal signalling pathways in mouse models of the NF1 cancer predisposition syndrome.
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Transformação Celular Neoplásica/genética , Genes da Neurofibromatose 1 , Mutação , Neurofibromina 1/genética , Neurônios/metabolismo , Glioma do Nervo Óptico/genética , Glioma do Nervo Óptico/patologia , Animais , Astrocitoma/genética , Astrocitoma/patologia , Moléculas de Adesão Celular Neuronais/deficiência , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Transformação Celular Neoplásica/efeitos da radiação , Feminino , Mutação em Linhagem Germinativa , Humanos , Masculino , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/efeitos da radiação , Nervo Óptico/citologia , Nervo Óptico/efeitos da radiação , Estimulação Luminosa , Retina/citologia , Retina/efeitos da radiaçãoRESUMO
Retinal ganglion cells (RGCs) are heterogeneous projection neurons that convey distinct visual features from the retina to brain. Here, we present a high-throughput in vivo RGC activity assay in response to light stimulation using noninvasive Ca2+ imaging of thousands of RGCs simultaneously in living mice. Population and single-cell analyses of longitudinal RGC Ca2+ imaging reveal distinct functional responses of RGCs and unprecedented individual RGC activity conversions during traumatic and glaucomatous degeneration. This study establishes a foundation for future in vivo RGC function classifications and longitudinal activity evaluations using more advanced imaging techniques and visual stimuli under normal, disease, and neural repair conditions. These analyses can be performed at both the population and single-cell levels using temporal and spatial information, which will be invaluable for understanding RGC pathophysiology and identifying functional biomarkers for diverse optic neuropathies.
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Glaucoma , Células Ganglionares da Retina , Animais , Camundongos , Diagnóstico por Imagem , Retina , Glaucoma/diagnóstico por imagem , EncéfaloRESUMO
Loss of retinal ganglion cells (RGCs) is central to the pathogenesis of optic neuropathies such as glaucoma. Increased RGC cAMP signaling is neuroprotective. We have shown that displacement of the cAMP-specific phosphodiesterase PDE4D3 from an RGC perinuclear compartment by expression of the modified PDE4D3 N-terminal peptide 4D3(E) increases perinuclear cAMP and protein kinase A activity in cultured neurons and in vivo RGC survival after optic nerve crush (ONC) injury. To explore mechanisms by which PDE4D3 displacement promotes neuroprotection, in this study mice intravitreally injected with an adeno-associated virus to express an mCherry-tagged 4D3(E) peptide were subjected to ONC injury and analyzed by single cell RNA-sequencing (scRNA-seq). 4D3(E)-mCherry expression was associated with an attenuation of injury-induced changes in gene expression, thereby supporting the hypothesis that enhanced perinuclear PKA signaling promotes neuroprotective RGC gene expression.
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Patients with inherited retinal dystrophies (IRDs) were recruited from two understudied populations: Mexico and Pakistan as well as a third well-studied population of European Americans to define the genetic architecture of IRD by performing whole-genome sequencing (WGS). Whole-genome analysis was performed on 409 individuals from 108 unrelated pedigrees with IRDs. All patients underwent an ophthalmic evaluation to establish the retinal phenotype. Although the 108 pedigrees in this study had previously been examined for mutations in known IRD genes using a wide range of methodologies including targeted gene(s) or mutation(s) screening, linkage analysis and exome sequencing, the gene mutations responsible for IRD in these 108 pedigrees were not determined. WGS was performed on these pedigrees using Illumina X10 at a minimum of 30X depth. The sequence reads were mapped against hg19 followed by variant calling using GATK. The genome variants were annotated using SnpEff, PolyPhen2, and CADD score; the structural variants (SVs) were called using GenomeSTRiP and LUMPY. We identified potential causative sequence alterations in 61 pedigrees (57%), including 39 novel and 54 reported variants in IRD genes. For 57 of these pedigrees the observed genotype was consistent with the initial clinical diagnosis, the remaining 4 had the clinical diagnosis reclassified based on our findings. In seven pedigrees (12%) we observed atypical causal variants, i.e. unexpected genotype(s), including 4 pedigrees with causal variants in more than one IRD gene within all affected family members, one pedigree with intrafamilial genetic heterogeneity (different affected family members carrying causal variants in different IRD genes), one pedigree carrying a dominant causative variant present in pseudo-recessive form due to consanguinity and one pedigree with a de-novo variant in the affected family member. Combined atypical and large structural variants contributed to about 20% of cases. Among the novel mutations, 75% were detected in Mexican and 50% found in European American pedigrees and have not been reported in any other population while only 20% were detected in Pakistani pedigrees and were not previously reported. The remaining novel IRD causative variants were listed in gnomAD but were found to be very rare and population specific. Mutations in known IRD associated genes contributed to pathology in 63% Mexican, 60% Pakistani and 45% European American pedigrees analyzed. Overall, contribution of known IRD gene variants to disease pathology in these three populations was similar to that observed in other populations worldwide. This study revealed a spectrum of mutations contributing to IRD in three populations, identified a large proportion of novel potentially causative variants that are specific to the corresponding population or not reported in gnomAD and shed light on the genetic architecture of IRD in these diverse global populations.
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Etnicidade/genética , Degeneração Retiniana/genética , Consanguinidade , Análise Mutacional de DNA/métodos , Exoma/genética , Proteínas do Olho/genética , Feminino , Estudos de Associação Genética/métodos , Ligação Genética/genética , Genótipo , Humanos , Masculino , México , Mutação/genética , Paquistão , Linhagem , Retina/patologia , Sequenciamento do Exoma/métodos , Sequenciamento Completo do Genoma/métodosRESUMO
Retinal ganglion cells (RGCs) die after optic nerve trauma or in degenerative disease. However, acute changes in protein expression that may regulate RGC response to injury are not fully understood, and detailed methods to quantify new protein synthesis have not been tested. Here, we develop and apply a new in vivo quantitative measure of newly synthesized proteins to examine changes occurring in the retina after optic nerve injury. Azidohomoalanine, a noncanonical amino acid, was injected intravitreally into the eyes of rodents of either sex with or without optic nerve injury. Isotope variants of biotin-alkyne were used for quantitative BONCAT (QBONCAT) mass spectrometry, allowing identification of protein synthesis and transport rate changes in more than 1000 proteins at 1 or 5 d after optic nerve injury. In vitro screening showed several newly synthesized proteins regulate axon outgrowth in primary neurons in vitro This novel approach to targeted quantification of newly synthesized proteins after injury uncovers a dynamic translational response within broader proteostasis regulation and enhances our understanding of the cellular response to injury.SIGNIFICANCE STATEMENT Optic nerve injury results in death and degeneration of retinal ganglion cells and their axons. The specific cellular response to injury, including changes in new protein synthesis, is obscured by existing proteins and protein degradation. In this study, we introduce QBONCAT to isolate and quantify acute protein synthesis and subsequent transport between cellular compartments. We identify novel candidate protein effectors of the regenerative response and uncover their regulation of axon growth in vitro, validating the utility of QBONCAT for the discovery of novel regulatory and therapeutic candidates after optic nerve injury.
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Traumatismos do Nervo Óptico , Axônios/metabolismo , Humanos , Regeneração Nervosa/fisiologia , Traumatismos do Nervo Óptico/metabolismo , Retina/metabolismo , Células Ganglionares da Retina/metabolismoRESUMO
PURPOSE: To evaluate associations of patient characteristics with United States eye care use and likelihood of blindness. DESIGN: Retrospective observational study. PARTICIPANTS: Patients (19 546 016) with 2018 visual acuity (VA) records in the American Academy of Ophthalmology's IRIS® Registry (Intelligent Research in Sight). METHODS: Legal blindness (20/200 or worse) and visual impairment (VI; worse than 20/40) were identified from corrected distance acuity in the better-seeing eye and stratified by patient characteristics. Multivariable logistic regressions evaluated associations with blindness and VI. Blindness was mapped by state and compared with population characteristics. Eye care use was analyzed by comparing population demographics with United States Census estimates and proportional demographic representation among blind patients versus a nationally representative US population sample (National Health and Nutritional Examination Survey [NHANES]). MAIN OUTCOME MEASURES: Prevalence and odds ratios for VI and blindness; proportional representation in the IRIS® Registry, Census, and NHANES by patient demographics. RESULTS: Visual impairment was present in 6.98% (n = 1 364 935) and blindness in 0.98% (n = 190 817) of IRIS patients. Adjusted odds of blindness were highest among patients ≥ 85 years old (odds ratio [OR], 11.85; 95% confidence interval [CI], 10.33-13.59 vs. those 0-17 years old). Blindness also was associated positively with rural location and Medicaid, Medicare, or no insurance vs. commercial insurance. Hispanic (OR, 1.59; 95% CI, 1.46-1.74) and Black (OR, 1.73; 95% CI, 1.63-1.84) patients showed a higher odds of blindness versus White non-Hispanic patients. Proportional representation in IRIS Registry relative to the Census was higher for White than Hispanic (2- to 4-fold) or Black (11%-85%) patients (P < 0.001). Blindness overall was less prevalent in NHANES than IRIS Registry; however, prevalence in adults aged 60+ was lowest among Black participants in the NHANES (0.54%) and second highest among comparable Black adults in IRIS (1.57%). CONCLUSIONS: Legal blindness from low VA was present in 0.98% of IRIS patients and associated with rural location, public or no insurance, and older age. Compared with US Census estimates, minorities may be underrepresented among ophthalmology patients, and compared with NHANES population estimates, Black individuals may be overrepresented among blind IRIS Registry patients. These findings provide a snapshot of US ophthalmic care and highlight the need for initiatives to address disparities in use and blindness. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.
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PIEZO1 and PIEZO2 are mechanosensitive ion channels that regulate many important physiological processes including vascular blood flow, touch, and proprioception. As the eye is subject to mechanical stress and is highly perfused, these channels may play important roles in ocular function and intraocular pressure regulation. PIEZO channel expression in the eye has not been well defined, in part due to difficulties in validating available antibodies against PIEZO1 and PIEZO2 in ocular tissues. It is also unclear if PIEZO1 and PIEZO2 are differentially expressed. To address these questions, we used single-molecule fluorescence in situ hybridization (smFISH) together with transgenic reporter mice expressing PIEZO fusion proteins under the control of their endogenous promoters to compare the expression and localization of PIEZO1 and PIEZO2 in mouse ocular tissues relevant to glaucoma. We detected both PIEZO1 and PIEZO2 expression in the trabecular meshwork, ciliary body, and in the ganglion cell layer (GCL) of the retina. Piezo1 mRNA was more abundantly expressed than Piezo2 mRNA in these ocular tissues. Piezo1 but not Piezo2 mRNA was detected in the inner nuclear layer and outer nuclear layer of the retina. Our results suggest that PIEZO1 and PIEZO2 are differentially expressed and may have distinct roles as mechanosensors in glaucoma-relevant ocular tissues.
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Glaucoma , Canais Iônicos , Animais , Camundongos , Glaucoma/genética , Hibridização in Situ Fluorescente , Canais Iônicos/metabolismo , Mecanotransdução Celular , Camundongos Transgênicos , RNA Mensageiro/genéticaRESUMO
cAMP signaling is known to be critical in neuronal survival and axon growth. Increasingly the subcellular compartmentation of cAMP signaling has been appreciated, but outside of dendritic synaptic regulation, few cAMP compartments have been defined in terms of molecular composition or function in neurons. Specificity in cAMP signaling is conferred in large part by A-kinase anchoring proteins (AKAPs) that localize protein kinase A and other signaling enzymes to discrete intracellular compartments. We now reveal that cAMP signaling within a perinuclear neuronal compartment organized by the large multivalent scaffold protein mAKAPα promotes neuronal survival and axon growth. mAKAPα signalosome function is explored using new molecular tools designed to specifically alter local cAMP levels as studied by live-cell FRET imaging. In addition, enhancement of mAKAPα-associated cAMP signaling by isoform-specific displacement of bound phosphodiesterase is demonstrated to increase retinal ganglion cell survival in vivo in mice of both sexes following optic nerve crush injury. These findings define a novel neuronal compartment that confers cAMP regulation of neuroprotection and axon growth and that may be therapeutically targeted in disease.SIGNIFICANCE STATEMENT cAMP is a second messenger responsible for the regulation of diverse cellular processes including neuronal neurite extension and survival following injury. Signal transduction by cAMP is highly compartmentalized in large part because of the formation of discrete, localized multimolecular signaling complexes by A-kinase anchoring proteins. Although the concept of cAMP compartmentation is well established, the function and identity of these compartments remain poorly understood in neurons. In this study, we provide evidence for a neuronal perinuclear cAMP compartment organized by the scaffold protein mAKAPα that is necessary and sufficient for the induction of neurite outgrowth in vitro and for the survival of retinal ganglion cells in vivo following optic nerve injury.
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Orientação de Axônios , AMP Cíclico/metabolismo , Células Ganglionares da Retina/metabolismo , Transdução de Sinais , Proteínas de Ancoragem à Quinase A/metabolismo , Animais , Axônios/metabolismo , Axônios/fisiologia , Células COS , Células Cultivadas , Chlorocebus aethiops , Feminino , Transferência Ressonante de Energia de Fluorescência , Masculino , Camundongos , Diester Fosfórico Hidrolases/metabolismo , Ratos , Ratos Sprague-Dawley , Células Ganglionares da Retina/citologia , Células Ganglionares da Retina/fisiologiaRESUMO
Adipose tissue-derived "stem cells" have been increasingly used by "stem-cell clinics" in the United States and elsewhere to treat a variety of disorders. We evaluated three patients in whom severe bilateral visual loss developed after they received intravitreal injections of autologous adipose tissue-derived "stem cells" at one such clinic in the United States. In these three patients, the last documented visual acuity on the Snellen eye chart before the injection ranged from 20/30 to 20/200. The patients' severe visual loss after the injection was associated with ocular hypertension, hemorrhagic retinopathy, vitreous hemorrhage, combined traction and rhegmatogenous retinal detachment, or lens dislocation. After 1 year, the patients' visual acuity ranged from 20/200 to no light perception.
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Tecido Adiposo/citologia , Degeneração Macular/terapia , Transplante de Células-Tronco/efeitos adversos , Transtornos da Visão/etiologia , Tecido Adiposo/transplante , Idoso , Idoso de 80 Anos ou mais , Cegueira/etiologia , Feminino , Humanos , Injeções , Descolamento Retiniano/etiologia , Transplante Autólogo/efeitos adversos , Acuidade VisualRESUMO
PURPOSE: The photopic negative response (PhNR) correlates with ganglion cell function and has previously been examined as an indicator of glaucomatous optic nerve damage. However, it is a prolonged response that is measured against baseline, and its clinical utility has been limited by extensive variability, poor repeatability, and baseline instability. We have observed a distinct brief negative wave ("N-wave") commonly present within the slow PhNR trough, which may provide practical and analytic advantages as a clinical measure. METHODS: We reviewed data from an interventional trial of 59 glaucoma patients who had 4 exams over an 8-month period. The PhNR was recorded with standard ISCEV stimuli (1 Hz and in some cases 4 Hz stimulation), and N-waves were measured manually, relative to return to baseline. RESULTS: N-waves, when present, could be measured easily despite shifting baselines and a degree of background noise. The PhNR median amplitude centered around 18 µV, while the N-wave median centered around 7 µV, with a distribution of responses skewed toward low or zero amplitudes. CONCLUSIONS: The N-wave appears to be a component of the longer PhNR, though its exact origin and significance remain unclear. As a rapid waveform that is independent of baseline, the N-wave is in many ways easier to measure accurately than the slower PhNR, which is highly dependent on baseline stability. The N-wave may prove useful clinically if further studies can optimize its stimulation, show its behavior in normal individuals and find correlation with markers of optic nerve disease.
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Visão de Cores/fisiologia , Glaucoma/fisiopatologia , Doenças do Nervo Óptico/fisiopatologia , Retina/fisiopatologia , Células Ganglionares da Retina/fisiologia , Administração Oftálmica , Adulto , Idoso , Idoso de 80 Anos ou mais , Método Duplo-Cego , Eletrorretinografia , Feminino , Glaucoma/tratamento farmacológico , Humanos , Masculino , Pessoa de Meia-Idade , Fator de Crescimento Neural/uso terapêutico , Soluções Oftálmicas , Doenças do Nervo Óptico/tratamento farmacológico , Estimulação Luminosa , Estudos Prospectivos , Proteínas Recombinantes , Adulto JovemRESUMO
PURPOSE: This study examined whether short-term use of topical nonsteroidal anti-inflammatory drug (NSAID) or steroid therapy affected the efficacy of selective laser trabeculoplasty (SLT). DESIGN: Double-masked, randomized, placebo-controlled, dual-center, multisurgeon trial. PARTICIPANTS: Patients older than 18 years with intraocular pressure (IOP) of more than 18 mmHg for whom the clinician decided SLT was the appropriately indicated therapy were randomized to 1 of 3 groups in a ratio of 1:1:1 as follows: ketorolac 0.5%, prednisolone 1%, or saline tears. METHODS: After SLT, patients randomized into each group were instructed to use an unmarked drop 4 times daily starting the day of SLT and continuing for 4 additional days. The Kruskal-Wallis test and Wilcoxon rank-sum test were used for continuous variables when comparing 2 or 3 treatment groups, respectively. The Fisher exact test was used for categorical variables. MAIN OUTCOME MEASURES: The primary outcome of this study was IOP at 12 weeks. Secondary outcome measures included IOP at 1 and 6 weeks, patient-reported pain, and detectable anterior chamber inflammation. RESULTS: Ninety-six eyes of 85 patients fit inclusion criteria and were enrolled between the 2 sites. The NSAID, steroid, and placebo groups were similar in baseline demographics and baseline IOP (mean, 23.3±3.9 mmHg; P = 0.57). There was no statistically significant difference in IOP decrease among groups at week 6. Both the NSAID and steroid groups showed a statistically significantly greater decrease in IOP at week 12 compared with the placebo group (mean, -6.2±3.1 mmHg, -5.2±2.7 mmHg, and -3±4.3 mmHg, respectively; P = 0.02 [analysis of variance] and P = 0.002 [t test] for NSAID vs. placebo groups; P = 0.02 for steroid vs. placebo groups). CONCLUSIONS: Significantly better IOP reduction at 12 weeks was measured in eyes treated with steroid or NSAID drops after SLT. Short-term postoperative use of NSAID or steroid drops may improve IOP reduction after SLT. Longer-term follow-up studies are indicated.
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Anti-Inflamatórios não Esteroides/uso terapêutico , Glaucoma de Ângulo Aberto/cirurgia , Glucocorticoides/uso terapêutico , Cetorolaco/uso terapêutico , Terapia a Laser/métodos , Prednisolona/uso terapêutico , Trabeculectomia/métodos , Administração Oftálmica , Idoso , Método Duplo-Cego , Feminino , Glaucoma de Ângulo Aberto/fisiopatologia , Humanos , Pressão Intraocular/fisiologia , Masculino , Pessoa de Meia-Idade , Hipertensão Ocular/tratamento farmacológico , Hipertensão Ocular/fisiopatologia , Soluções Oftálmicas , Período Pós-Operatório , Tonometria Ocular , Malha Trabecular/cirurgia , Resultado do TratamentoRESUMO
Neurons in the adult mammalian CNS decrease in intrinsic axon growth capacity during development in concert with changes in Krüppel-like transcription factors (KLFs). KLFs regulate axon growth in CNS neurons including retinal ganglion cells (RGCs). Here, we found that knock-down of KLF9, an axon growth suppressor that is normally upregulated 250-fold in RGC development, promotes long-distance optic nerve regeneration in adult rats of both sexes. We identified a novel binding partner, MAPK10/JNK3 kinase, and found that JNK3 (c-Jun N-terminal kinase 3) is critical for KLF9's axon-growth-suppressive activity. Interfering with a JNK3-binding domain or mutating two newly discovered serine phosphorylation acceptor sites, Ser106 and Ser110, effectively abolished KLF9's neurite growth suppression in vitro and promoted axon regeneration in vivo These findings demonstrate a novel, physiologic role for the interaction of KLF9 and JNK3 in regenerative failure in the optic nerve and suggest new therapeutic strategies to promote axon regeneration in the adult CNS.SIGNIFICANCE STATEMENT Injured CNS nerves fail to regenerate spontaneously. Promoting intrinsic axon growth capacity has been a major challenge in the field. Here, we demonstrate that knocking down Krüppel-like transcription factor 9 (KLF9) via shRNA promotes long-distance axon regeneration after optic nerve injury and uncover a novel and important KLF9-JNK3 interaction that contributes to axon growth suppression in vitro and regenerative failure in vivo These studies suggest potential therapeutic approaches to promote axon regeneration in injury and other degenerative diseases in the adult CNS.
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Axônios/fisiologia , Encéfalo/fisiologia , Fatores de Transcrição Kruppel-Like/metabolismo , Proteína Quinase 10 Ativada por Mitógeno/metabolismo , Regeneração Nervosa/fisiologia , Fatores Etários , Animais , Sequência de Bases , Células Cultivadas , Sistema Nervoso Central/fisiologia , Feminino , Fatores de Transcrição Kruppel-Like/deficiência , Fatores de Transcrição Kruppel-Like/genética , Masculino , Camundongos , Proteína Quinase 10 Ativada por Mitógeno/genética , Traumatismos do Nervo Óptico/genética , Traumatismos do Nervo Óptico/metabolismo , Técnicas de Cultura de Órgãos , Ligação Proteica/fisiologia , Ratos , Células Ganglionares da Retina/fisiologiaRESUMO
What pathways specify retinal ganglion cell (RGC) fate in the developing retina? Here we report on mechanisms by which a molecular pathway involving Sox4/Sox11 is required for RGC differentiation and for optic nerve formation in mice in vivo, and is sufficient to differentiate human induced pluripotent stem cells into electrophysiologically active RGCs. These data place Sox4 downstream of RE1 silencing transcription factor in regulating RGC fate, and further describe a newly identified, Sox4-regulated site for post-translational modification with small ubiquitin-related modifier (SUMOylation) in Sox11, which suppresses Sox11's nuclear localization and its ability to promote RGC differentiation, providing a mechanism for the SoxC familial compensation observed here and elsewhere in the nervous system. These data define novel regulatory mechanisms for this SoxC molecular network, and suggest pro-RGC molecular approaches for cell replacement-based therapies for glaucoma and other optic neuropathies.SIGNIFICANCE STATEMENT Glaucoma is the most common cause of blindness worldwide and, along with other optic neuropathies, is characterized by loss of retinal ganglion cells (RGCs). Unfortunately, vision and RGC loss are irreversible, and lead to bilateral blindness in â¼14% of all diagnosed patients. Differentiated and transplanted RGC-like cells derived from stem cells have the potential to replace neurons that have already been lost and thereby to restore visual function. These data uncover new mechanisms of retinal progenitor cell (RPC)-to-RGC and human stem cell-to-RGC fate specification, and take a significant step toward understanding neuronal and retinal development and ultimately cell-transplant therapy.
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Envelhecimento/fisiologia , Redes Reguladoras de Genes/fisiologia , Células Ganglionares da Retina/fisiologia , Fatores de Transcrição SOXC/metabolismo , Ativação Transcricional/fisiologia , Vias Visuais/fisiologia , Animais , Células Cultivadas , Retroalimentação Fisiológica/fisiologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Masculino , Camundongos , Ratos Sprague-DawleyRESUMO
The lack of intravital imaging of axonal transport of mitochondria in the mammalian CNS precludes characterization of the dynamics of axonal transport of mitochondria in the diseased and aged mammalian CNS. Glaucoma, the most common neurodegenerative eye disease, is characterized by axon degeneration and the death of retinal ganglion cells (RGCs) and by an age-related increase in incidence. RGC death is hypothesized to result from disturbances in axonal transport and in mitochondrial function. Here we report minimally invasive intravital multiphoton imaging of anesthetized mouse RGCs through the sclera that provides sequential time-lapse images of mitochondria transported in a single axon with submicrometer resolution. Unlike findings from explants, we show that the axonal transport of mitochondria is highly dynamic in the mammalian CNS in vivo under physiological conditions. Furthermore, in the early stage of glaucoma modeled in adult (4-mo-old) mice, the number of transported mitochondria decreases before RGC death, although transport does not shorten. However, with increasing age up to 23-25 mo, mitochondrial transport (duration, distance, and duty cycle) shortens. In axons, mitochondria-free regions increase and lengths of transported mitochondria decrease with aging, although totally organized transport patterns are preserved in old (23- to 25-mo-old) mice. Moreover, axonal transport of mitochondria is more vulnerable to glaucomatous insults in old mice than in adult mice. These mitochondrial changes with aging may underlie the age-related increase in glaucoma incidence. Our method is useful for characterizing the dynamics of axonal transport of mitochondria and may be applied to other submicrometer structures in the diseased and aged mammalian CNS in vivo.
Assuntos
Envelhecimento , Transporte Axonal/fisiologia , Sistema Nervoso Central/patologia , Sistema Nervoso Central/fisiologia , Mitocôndrias/fisiologia , Células Ganglionares da Retina/fisiologia , Animais , Axônios/fisiologia , Transporte Biológico , Modelos Animais de Doenças , Feminino , Glaucoma/patologia , Glaucoma/fisiopatologia , Imageamento Tridimensional , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Nervo Óptico/patologia , Fótons , Retina/citologia , Esclera/fisiopatologia , Fatores de TempoRESUMO
Regenerative medicine holds great promise for the treatment of degenerative retinal disorders. Krüppel-like factors (KLFs) are transcription factors that have recently emerged as key tools in regenerative medicine because some of them can function as epigenetic reprogrammers in stem cell biology. Here, we show that KLF16, one of the least understood members of this family, is a POU4F2 independent transcription factor in retinal ganglion cells (RGCs) as early as embryonic day 15. When overexpressed, KLF16 inhibits RGC neurite outgrowth and enhances RGC growth cone collapse in response to exogenous ephrinA5 ligands. Ephrin/EPH signaling regulates RGC connectivity. The EphA5 promoter contains multiple GC- and GT-rich KLF-binding sites, which, as shown by ChIP-assays, bind KLF16 in vivo In electrophoretic mobility shift assays, KLF16 binds specifically to a single KLF site near the EphA5 transcription start site that is required for KLF16 transactivation. Interestingly, methylation of only six of 98 CpG dinucleotides within the EphA5 promoter blocks its transactivation by KLF16 but enables transactivation by KLF2 and KLF15. These data demonstrate a role for KLF16 in regulation of RGC neurite outgrowth and as a methylation-sensitive transcriptional regulator of EphA5 expression. Together, these data identify differential low level methylation as a novel mechanism for regulating KLF16-mediated EphA5 expression across the retina. Because of the critical role of ephrin/EPH signaling in patterning RGC connectivity, understanding the role of KLFs in regulating neurite outgrowth and Eph receptor expression will be vital for successful restoration of functional vision through optic nerve regenerative therapies.
Assuntos
Fatores de Transcrição Kruppel-Like/metabolismo , Neuritos/metabolismo , Receptor EphA5/biossíntese , Elementos de Resposta/fisiologia , Células Ganglionares da Retina/metabolismo , Transdução de Sinais/fisiologia , Ativação Transcricional/fisiologia , Animais , Metilação de DNA , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Camundongos Knockout , Receptor EphA5/genética , Células Ganglionares da Retina/citologia , Transcrição Gênica/fisiologiaRESUMO
Intraocular pressure (IOP)-lowering ophthalmic solutions that inhibit Rho-associated protein kinases (Rock) and norepinephrine transporters (Net) are currently under clinical evaluation. Here we evaluate topical application of one such drug for its effects on retinal ganglion cell (RGC) survival and axon regeneration after optic nerve crush injury. We performed unilateral optic nerve crush on young rats (P18) and topically applied Rock/Net inhibitor AR-13324 or placebo 3 times a day for 14 days. IOP was measured starting 3 days before and up to 9 days after injury. On day 12, cholera toxin B (CTB) was injected intravitreally to trace optic nerve regeneration. On day 14, retinas and optic nerves were collected. The retinas were flat-mounted and stained with RBPMS to quantify RGC survival and the optic nerves were sectioned for optic nerve axon quantification using fluorescent and confocal microscopy. Rock phosphorylation targets implicated in axon growth including cofilin and LIMK were examined by fluorescence microscopy and quantitative western blotting. AR-13324 lowered IOP as expected. RGC survival and optic nerve axon regeneration were significantly higher with Rock/Net inhibitor treatment compared with placebo. Furthermore, topical therapy decreased Rock target protein phosphorylation in the retinas and proximal optic nerves. These data suggest that topical administration of a Rock/Net inhibitor promotes RGC survival and regeneration after optic nerve injury, with associated molecular changes indicative of posterior drug activity. Coordinated IOP lowering and neuroprotective or regenerative effects may be advantageous in the treatment of patients with glaucoma.
Assuntos
Axônios/fisiologia , Benzoatos/administração & dosagem , Regeneração Nervosa/efeitos dos fármacos , Proteínas da Membrana Plasmática de Transporte de Norepinefrina/antagonistas & inibidores , Traumatismos do Nervo Óptico/tratamento farmacológico , Células Ganglionares da Retina/citologia , beta-Alanina/análogos & derivados , Quinases Associadas a rho/antagonistas & inibidores , Administração Tópica , Animais , Western Blotting , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Glaucoma/tratamento farmacológico , Glaucoma/fisiopatologia , Imuno-Histoquímica , Pressão Intraocular/efeitos dos fármacos , Masculino , Compressão Nervosa , Soluções Oftálmicas , Nervo Óptico/fisiologia , Traumatismos do Nervo Óptico/fisiopatologia , Fosforilação , Ratos , Ratos Sprague-Dawley , Tonometria Ocular , beta-Alanina/administração & dosagemRESUMO
Set-ß protein plays different roles in neurons, but the diversity of Set-ß neuronal isoforms and their functions have not been characterized. The expression and subcellular localization of Set-ß are altered in Alzheimer disease, cleavage of Set-ß leads to neuronal death after stroke, and the full-length Set-ß regulates retinal ganglion cell (RGC) and hippocampal neuron axon growth and regeneration in a subcellular localization-dependent manner. Here we used various biochemical approaches to investigate Set-ß isoforms and their role in the CNS, using the same type of neurons, RGCs, across studies. We found multiple alternatively spliced isoforms expressed from the Set locus in purified RGCs. Set transcripts containing the Set-ß-specific exon were the most highly expressed isoforms. We also identified a novel, alternatively spliced Set-ß transcript lacking the nuclear localization signal and demonstrated that the full-length (â¼39-kDa) Set-ß is localized predominantly in the nucleus, whereas a shorter (â¼25-kDa) Set-ß isoform is localized predominantly in the cytoplasm. Finally, we show that an N-terminal Set-ß cleavage product can induce neuronal death.
Assuntos
Processamento Alternativo/genética , Apoptose , Proteínas de Transporte/metabolismo , Neurônios/patologia , Proteínas Nucleares/metabolismo , Proteínas Oncogênicas/metabolismo , Células Ganglionares da Retina/patologia , Animais , Animais Recém-Nascidos , Western Blotting , Proteínas de Transporte/genética , Proliferação de Células , Células Cultivadas , Proteínas de Ligação a DNA , Imunofluorescência , Chaperonas de Histonas , Técnicas Imunoenzimáticas , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Proteínas Nucleares/genética , Proteínas Oncogênicas/genética , Isoformas de Proteínas , RNA Mensageiro/genética , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real , Células Ganglionares da Retina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Stem cells hold promise for treating a wide variety of diseases, including degenerative disorders of the eye. The eye is an ideal organ for stem cell therapy because of its relative immunological privilege, surgical accessibility, and its being a self-contained system. The eye also has many potential target diseases amenable to stem cell-based treatment, such as corneal limbal stem cell deficiency, glaucoma, age-related macular degeneration (AMD), and retinitis pigmentosa (RP). Among them, AMD and glaucoma are the two most common diseases, affecting over 200 million people worldwide. Recent results on the clinical trial of retinal pigment epithelial (RPE) cells from human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) in treating dry AMD and Stargardt's disease in the US, Japan, England, and China have generated great excitement and hope. This marks the beginning of the ocular stem cell therapy era. The recent Zhongshan Ophthalmic Center Ocular Stem Cell Symposium discussed the potential applications of various stem cell types in stem cell-based therapies, drug discoveries and tissue engineering for treating ocular diseases.