Dr. Jeffrey Goldberg is Professor and Chair of Ophthalmology at the Byers Eye Institute at Stanford University, and a member of the National Academy of Medicine. His clinical effort is focused on patients in need of medical or surgical intervention for glaucoma and other retinal and optic nerve diseases, as well as cataract. His research is directed at neuroprotection and regeneration of retinal ganglion cells and the optic nerve, a major unmet need in glaucoma and other optic neuropathies, and his laboratory is developing novel stem cell and nanotherapeutics approaches for eye repair.
Dr. Goldberg received his B.S. magna cum laude from Yale University, and his M.D. and Ph.D. from Stanford University where he made significant discoveries about the failure of optic nerve regeneration. He did his clinical training in ophthalmology and then in glaucoma at the Bascom Palmer Eye Institute, and was awarded a fellowship from the Heed Foundation. He was named the 2010 Scientist of the Year by the Hope For Vision foundation, and received the Cogan award from the Association for Research in Vision and Ophthalmology in 2012. He was elected in 2010 to the American Society of Clinical Investigation, an honorary society of physician scientists. He directs an NIH-funded research laboratory and has developed significant expertise with implementing FDA IND clinical trials for optic nerve neuroprotection and regeneration. His goal is to translate scientific discoveries to patient therapies. A partial list of clinical trials can be found on the department webpage, https://med.stanford.edu/ophthalmology/research/clinical_trials.html.
Chair of Ophthlmology, Stanford University (2015 - Present)
Fellowship: Bascom Palmer Eye Institute (2010) FL
Residency: Bascom Palmer Eye Institute (2008) FL
Board Certification: American Board of Ophthalmology, Ophthalmology (2009)
Internship: Santa Clara Valley Medical Center (2004) CA
Medical Education: Stanford School of Medicine (2003) CA
Fellowship, Bascom Palmer Eye Institute, Glaucoma (2010)
PhD, Stanford University, Neurosciences (2003)
MD, Stanford University, Medicine (2003)
Current Research and Scholarly Interests
Lab research on molecular mechanisms of survival and regeneration in the visual system; retinal development and stem cell biology; nanoparticles and tissue engineering. Clinical trials in imaging, biomarker development, and neuroprotection and vision restoration in glaucoma and other neurodegenerative diseases.
Dual Intravitreal Implantation of NT-501 Encapsulated Cell Therapy for Glaucoma
To determine the safety and efficacy over 24 months of dual NT-501 CNTF encapsulated cell therapy (ECT) on visual impairment related to glaucoma.
- Independent Studies (6)
Graduate and Fellowship Programs
Post-translational modification of Sox11 regulates RGC survival and axon regeneration.
The failure of adult CNS neurons to survive and regenerate their axons after injury or in neurodegenerative disease remains a major target for basic and clinical neuroscience. Recent data demonstrated in the adult mouse that exogenous expression of Sry-related high-mobility-box 11 (Sox11) promotes optic nerve regeneration after optic nerve injury, but exacerbates the death of a subset of retinal ganglion cells, alpha-RGCs. During development, Sox11 is required for RGC differentiation from retinal progenitor cells (RPCs), and we found that mutation of a single residue to prevent sumoylation at lysine 91 (K91) increased nuclear localization and RGC differentiation in vitro Here we explored whether this Sox11 manipulation similarly has stronger effects on RGC survival and optic nerve regeneration. In vitro, we found that non-SUMOylatable Sox11K91A leads to RGC death and suppresses axon outgrowth in primary neurons. We furthermore found that Sox11K91A more strongly promotes axon regeneration but also increases RGC death after optic nerve injury in vivo in adult mouse. RNA sequence data showed that Sox11 and Sox11K91A increase the expression of key signaling pathway genes associated with axon growth and regeneration but downregulated Spp1 and Opn4 expression in RGC cultures, consistent with negatively regulating the survival of alpha-RGCs and ipRGCs. Thus Sox11 and its sumoylation site at K91 regulate gene expression, survival and axon growth in RGCs and may be explored further as potential regenerative therapies for optic neuropathy.Significance Statement Sox11 expression promotes optic nerve regeneration but also increases RGC death after optic nerve injury. Here we demonstrate that mutation of a single SUMOylation site on Sox11 (Sox11K91A) leads to stronger effects in vivo RNA sequencing analysis reveals that Sox11 and Sox11K91A differentially regulate downstream gene expression related to axon growth and guidance. Understanding these effects of post-translational modification of Sox11 in regulating regeneration in vivo suggests a potent therapeutic strategy for vision restoration in optic neuropathies.
View details for DOI 10.1523/ENEURO.0358-20.2020
View details for PubMedID 33441400
Dual Specific Phosphatase 14 Deletion Rescues Retinal Ganglion Cells and Optic Nerve Axons after Experimental Anterior Ischemic Optic Neuropathy.
Current eye research
PURPOSE: Understanding molecular changes is essential for designing effective treatments for nonarteritic anterior ischemic optic neuropathy (AION), the most common acute optic neuropathy in adults older than 50years. We investigated changes in the mitogen-activated protein kinase (MAPK) pathway after experimental AION and focused on dual specificity phosphatase 14 (Dusp14), an atypical MAPK phosphatase that is downstream of Kruppel-like transcription factor (KLF) 9-mediated inhibition of retinal ganglion cell (RGC) survival and axonal regeneration.MATERIALS AND METHODS: We induced severe AION in a photochemical thrombosis model in adult C57BL/6 wild-type and Dusp14 knockout mice. For comparison, some studies were performed using an optic nerve crush model. We assessed changes in MAPK pathway molecules using Western blot and immunohistochemistry, measured retinal thickness using optical coherence tomography (OCT), and quantified RGCs and axons using histologic methods.RESULTS: Three days after severe AION, there was no change in the retinal protein levels of MAPK ERK1/2, phosphorylated-ERK1/2 (pERK1/2), downstream effector Elk-1 and phosphatase Dusp14 on Western blot. Western blot analysis of purified RGCs after a more severe model using optic nerve crush also showed no change in Dusp14 protein expression. Because of the known importance of the Dusp14 and MAPK pathway in RGCs, we examined changes after AION in Dusp14 knockout mice. Three days after AION, Dusp14 knockout mice had significantly increased pERK1/2+, Brn3A+ RGCs on immunohistochemistry. Three weeks after AION, Dusp14 knockout mice had significantly greater preservation of retinal thickness, increased number of Brn3A+ RGCs on whole mount preparation, and increased number of optic nerve axons compared with wild-type mice.CONCLUSIONS: Genetic deletion of Dusp14, a MAPK phosphatase important in KFL9-mediated inhibition of RGC survival, led to increased activation of MAPK ERK1/2 and greater RGC and axonal survival after experimental AION. Inhibiting Dusp14 or activating the MAPK pathway should be examined further as a potential therapeutic approach to treatment of AION.Abbreviations: AION: anterior ischemic optic neuropathy; Dusp14: dual specific phosphatase 14; ERK1/2: extracellular signal-regulated kinases 1/2; Elk-1: ETS Like-1 protein; GCC: ganglion cell complex; GCL: ganglion cell layer; inner nuclear layer; KO: knockout; MAPK: mitogen-activated phosphokinase; OCT: optical coherence tomography; RGC: retinal ganglion cell; RNFL: retinal nerve fiber layer.
View details for DOI 10.1080/02713683.2020.1826976
View details for PubMedID 33107352
Dynamics of Contrast Decrement and Increment Responses in Human Visual Cortex.
Translational vision science & technology
2020; 9 (10): 6
Purpose: The goal of the present experiments was to determine whether electrophysiologic response properties of the ON and OFF visual pathways observed in animal experimental models can be observed in humans.Methods: Steady-state visual evoked potentials (SSVEPs) were recorded in response to equivalent magnitude contrast increments and decrements presented within a probe-on-pedestal Westheimer sensitization paradigm. The probes were modulated with sawtooth temporal waveforms at a temporal frequency of 3 or 2.73 Hz. SSVEP response waveforms and response spectra for incremental and decremental stimuli were analyzed as a function of stimulus size and visual field location in 67 healthy adult participants.Results: SSVEPs recorded at the scalp differ between contrast decrements and increments of equal Weber contrast: SSVEP responses were larger in amplitude and shorter in latency for contrast decrements than for contrast increments. Both increment and decrement responses were larger for displays that were scaled for cortical magnification.Conclusions: In a fashion that parallels results from the early visual system of cats and monkeys, two key properties of ON versus OFF pathways found in single-unit recordings are recapitulated at the population level of activity that can be observed with scalp electrodes, allowing differential assessment of ON and OFF pathway activity in human.Translational Relevance: As data from preclinical models of visual pathway dysfunction point to differential damage to subtypes of retinal ganglion cells, this approach may be useful in future work on disease detection and treatment monitoring.
View details for DOI 10.1167/tvst.9.10.6
View details for PubMedID 32953246
Discovery and clinical translation of novel glaucoma biomarkers.
Progress in retinal and eye research
Glaucoma and other optic neuropathies are characterized by progressive dysfunction and loss of retinal ganglion cells and their axons. Given the high prevalence of glaucoma-related blindness and the availability of treatment options, improving the diagnosis and precise monitoring of progression in these conditions is paramount. Here we review recent progress in the development of novel biomarkers for glaucoma in the context of disease pathophysiology and we propose future steps for the field, including integration of exploratory biomarker outcomes into prospective therapeutic trials. We anticipate that, when validated, some of the novel glaucoma biomarkers discussed here will prove useful for clinical diagnosis and prediction of progression, as well as monitoring of clinical responses to standard and investigational therapies.
View details for DOI 10.1016/j.preteyeres.2020.100875
View details for PubMedID 32659431
An Open-Label Phase Ib Study to Evaluate Retinal Imaging After Short-term Use of the Balance Goggles System (BGS) in Patients with Glaucoma
ASSOC RESEARCH VISION OPHTHALMOLOGY INC. 2020
View details for Web of Science ID 000554528303047
Physiological and morphological development of human embryonic stem cell derived retinal ganglion cells
ASSOC RESEARCH VISION OPHTHALMOLOGY INC. 2020
View details for Web of Science ID 000554528302119
Transcription factor REST/NRSF regulates optic nerve regeneration and RGC survival
ASSOC RESEARCH VISION OPHTHALMOLOGY INC. 2020
View details for Web of Science ID 000554528300014
Retinal organoid differentiation and transplantatio
ASSOC RESEARCH VISION OPHTHALMOLOGY INC. 2020
View details for Web of Science ID 000554495705088
Optic Nerve Crush in Mice to Study Retinal Ganglion Cell Survival and Regeneration.
2020; 10 (6)
In diseases such as glaucoma, the failure of retinal ganglion cell (RGC) neurons to survive or regenerate their optic nerve axons underlies partial and, in some cases, complete vision loss. Optic nerve crush (ONC) serves as a useful model not only of traumatic optic neuropathy but also of glaucomatous injury, as it similarly induces RGC cell death and degeneration. Intravitreal injection of adeno-associated virus serotype 2 (AAV2) has been shown to specifically and efficiently transduce RGCs in vivo and has thus been proposed as an effective means of gene delivery for the treatment of glaucoma. Indeed, we and others routinely use AAV2 to study the mechanisms that promote neuroprotection and axon regeneration in RGCs following ONC. Herein, we describe a step-by-step protocol to assay RGC survival and regeneration in mice following AAV2-mediated transduction and ONC injury including 1) intravitreal injection of AAV2 viral vectors, 2) optic nerve crush, 3) cholera-toxin B (CTB) labeling of regenerating axons, 4) optic nerve clearing, 5) flat mount retina immunostaining, and 6) quantification of RGC survival and regeneration. In addition to providing all the materials and procedural details necessary to execute this protocol, we highlight its advantages over other similar published approaches and include useful tips to ensure its faithful reproduction in any modern laboratory.
View details for DOI 10.21769/BioProtoc.3559
View details for PubMedID 32368566
NONINFECTIOUS POSTERIOR UVEITIS BRINGING EMERGING TREATMENTS TO THE FOREFRONT
RETINA-THE JOURNAL OF RETINAL AND VITREOUS DISEASES
View details for Web of Science ID 000554613800001
Re: Groth et al.: SALT Trial: steroids after laser trabeculoplasty: impact of short-term anti-inflammatory treatment on selective laser trabeculoplasty efficacy (Ophthalmology. 2019;126:1511-1516) REPLY
2020; 127 (2): E17
View details for Web of Science ID 000508226400006
- Reply. Ophthalmology 2020; 127 (2): e17
The rapid N-wave as a potentially useful measure of the photopic negative response.
Documenta ophthalmologica. Advances in ophthalmology
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.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.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.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.
View details for DOI 10.1007/s10633-020-09769-w
View details for PubMedID 32507902
Multi-Omic Analyses of Growth Cones at Different Developmental Stages Provides Insight into Pathways in Adult Neuroregeneration.
2020; 23 (2): 100836
Growth cones (GCs) are structures associated with growing neurons. GC membrane expansion, which necessitates protein-lipid interactions, is critical to axonal elongation in development and in adult neuritogenesis. We present a multi-omic analysis that integrates proteomics and lipidomics data for the identification of GC pathways, cell phenotypes, and lipid-protein interactions, with an analytic platform to facilitate the visualization of these data. We combine lipidomic data from GC and adult axonal regeneration following optic nerve crush. Our results reveal significant molecular variability in GCs across developmental ages that aligns with the upregulation and downregulation of lipid metabolic processes and correlates with distinct changes in the lipid composition of GC plasmalemma. We find that these processes also define the transition into a growth-permissive state in the adult central nervous system. The insight derived from these analyses will aid in promoting adult regeneration and functional innervation in devastating neurodegenerative diseases.
View details for DOI 10.1016/j.isci.2020.100836
View details for PubMedID 32058951
View details for PubMedCentralID PMC6997871
MEF2 transcription factors differentially contribute to retinal ganglion cell loss after optic nerve injury.
2020; 15 (12): e0242884
Loss of retinal ganglion cells (RGCs) in optic neuropathies results in permanent partial or complete blindness. Myocyte enhancer factor 2 (MEF2) transcription factors have been shown to play a pivotal role in neuronal systems, and in particular MEF2A knockout was shown to enhance RGC survival after optic nerve crush injury. Here we expanded these prior data to study bi-allelic, tri-allelic and heterozygous allele deletion. We observed that deletion of all MEF2A, MEF2C, and MEF2D alleles had no effect on RGC survival during development. Our extended experiments suggest that the majority of the neuroprotective effect was conferred by complete deletion of MEF2A but that MEF2D knockout, although not sufficient to increase RGC survival on its own, increased the positive effect of MEF2A knockout. Conversely, MEF2A over-expression in wildtype mice worsened RGC survival after optic nerve crush. Interestingly, MEF2 transcription factors are regulated by post-translational modification, including by calcineurin-catalyzed dephosphorylation of MEF2A Ser-408 known to increase MEF2A-dependent transactivation in neurons. However, neither phospho-mimetic nor phospho-ablative mutation of MEF2A Ser-408 affected the ability of MEF2A to promote RGC death in vivo after optic nerve injury. Together these findings demonstrate that MEF2 gene expression opposes RGC survival following axon injury in a complex hierarchy, and further support the hypothesis that loss of or interference with MEF2A expression might be beneficial for RGC neuroprotection in diseases such as glaucoma and other optic neuropathies.
View details for DOI 10.1371/journal.pone.0242884
View details for PubMedID 33315889
The Retinal Ganglion Cell Transportome Identifies Proteins Transported to Axons and Presynaptic Compartments in the Visual System InVivo.
2019; 28 (7): 1935
The brain processes information and generates cognitive and motor outputs through functions of spatially organized proteins in different types of neurons. More complete knowledge of proteins and their distributions within neuronal compartments in intact circuits would help in the understanding of brain function. We used unbiased invivo protein labeling with intravitreal NHS-biotin for discovery and analysis of endogenous axonally transported proteins in the visual system using tandem mass spectrometric proteomics, biochemistry, and both light and electron microscopy. Purification and proteomic analysis of biotinylated peptides identified 1,000 proteins transported from retinal ganglion cells into the optic nerve and 575 biotinylated proteins recovered from presynaptic compartments of lateral geniculate nucleus and superior colliculus. Approximately 360 biotinylated proteins were differentially detected in the two retinal targets. This study characterizes axonally transported proteins in the healthy adult visual system by analyzing proteomes from multiple compartments of retinal ganglion cell projections in the intact brain.
View details for DOI 10.1016/j.celrep.2019.07.037
View details for PubMedID 31412257
- Regulation of Neuronal Survival and Axon Growth by a Perinuclear cAMP Compartment JOURNAL OF NEUROSCIENCE 2019; 39 (28): 5466–80
The Role of MEF2 transcription factors in retinal ganglion cell survival and axon regeneration
ASSOC RESEARCH VISION OPHTHALMOLOGY INC. 2019
View details for Web of Science ID 000488800703192
Dynamic Transcriptional Profiling of Reactive Muller Glia Following Retinal Injury
ASSOC RESEARCH VISION OPHTHALMOLOGY INC. 2019
View details for Web of Science ID 000488800702170
- Silicone oil-induced ocular hypertension and glaucomatous neurodegeneration in mouse ELIFE 2019; 8
Silicone oil-induced ocular hypertension and glaucomatous neurodegeneration in mouse.
Understanding the molecular mechanism of glaucoma and development of neuroprotectants are significantly hindered by the lack of a reliable animal model that accurately recapitulates human glaucoma. Here we sought to develop a mouse model for the secondary glaucoma that is often observed in humans after silicone oil (SO) blocks the pupil or migrates into the anterior chamber following vitreoretinal surgery. We observed significant intraocular pressure (IOP) elevation after intracameral injection of SO, and that SO removal allows IOP to return quickly to normal. This simple, inducible and reversible mouse ocular hypertension model shows dynamic changes of visual function that correlate with progressive RGC loss and axon degeneration. It may be applicable with only minor modifications to a range of animal species in which it will generate stable, robust IOP elevation and significant neurodegeneration that will facilitate selection of neuroprotectants and investigating the pathogenesis of ocular hypertension-induced glaucoma.
View details for PubMedID 31090540
Opposing Effects of Growth and Differentiation Factors in Cell-Fate Specification.
Current biology : CB
Following ocular trauma or in diseases such as glaucoma, irreversible vision loss is due to the death of retinal ganglion cell (RGC) neurons. Although strategies to replace these lost cells include stem cell replacement therapy, few differentiated stem cells turn into RGC-like neurons. Understanding the regulatory mechanisms of RGC differentiation in vivo may improve outcomes of cell transplantation by directing the fate of undifferentiated cells toward mature RGCs. Here, we report a new mechanism by which growth and differentiation factor-15 (GDF-15), a ligand in the transforming growth factor-beta (TGF-β) superfamily, strongly promotes RGC differentiation in the developing retina in vivo in rodent retinal progenitor cells (RPCs) and in human embryonic stem cells (hESCs). This effect is in direct contrast to the closely related ligand GDF-11, which suppresses RGC-fate specification. We find these opposing effects are due in part to GDF-15's ability to specifically suppress Smad-2, but not Smad-1, signaling induced by GDF-11, which can be recapitulated by pharmacologic or genetic blockade of Smad-2 in vivo to increase RGC specification. No other retinal cell types were affected by GDF-11 knockout, but a slight reduction in photoreceptor cells was observed by GDF-15 knockout in the developing retina in vivo. These data define a novel regulatory mechanism of GDFs' opposing effects and their relevance in RGC differentiation and suggest a potential approach for advancing ESC-to-RGC cell-based replacement therapies.
View details for DOI 10.1016/j.cub.2019.05.011
View details for PubMedID 31155355
Molecular Biomarkers for Glaucoma.
Current ophthalmology reports
2019; 7 (3): 171–76
Purpose of review: This article summarizes the current studies on molecular biomarkers with potential implications in diagnosis, prognosis, and response to treatment in patients with glaucoma.Recent findings: Important advances have occurred in the understanding of the pathogenesis of glaucomatous neurodegeneration. Protein biomarkers associated with inflammatory, neurodegenerative, and other molecular pathways have been described in glaucoma patients in tear film, aqueous fluid, vitreous fluid, and serum, however, we are still far from having a clear picture of the whole molecular network that relates to the disease and its implications in clinical use.Summary: Although more studies are needed, current and emerging molecular biomarkers candidates in glaucoma may eventually transition into clinical use and contribute to outline the concept of precision medicine and precision health in glaucoma.
View details for DOI 10.1007/s40135-019-00213-0
View details for PubMedID 33133776
Cell transplantation of retinal ganglion cells derived from hESCs.
Restorative neurology and neuroscience
Glaucoma, the number one cause of irreversible blindness, is characterized by the loss of retinal ganglion cells (RGCs), which do not regenerate in humans or mammals after cell death. Cell transplantation provides an opportunity to restore vision in glaucoma, or other optic neuropathies. Since transplanting primary RGCs from deceased donor tissues may not be feasible, stem cell-derived RGCs could provide a plausible alternative source of donor cells for transplant.We define a robust chemically defined protocol to differentiate human embryonic stem cells (hESCs) into RGC-like neurons.Human embryonic stem cell lines (H7-A81 and H9) and induced pluripotent stem cell (iPSC) were used for RGC differentiation. RGC immaturity was measured by calcium imaging against muscimol. Cell markers were detected by immunofluorescence staining and qRT-PCR. RGC-like cells were intravitreally injected to rat eye, and co-stained with RBPMS and human nuclei markers. All experiments were conducted at least three times independently. Data were analyzed by ANOVA with Tukey's test with P value of <0.05 considered statistically significant.We detected retinal progenitor markers Rx and Pax6 after 15 days of differentiation, and the expression of markers for RGC-specific differentiation (Brn3a and Brn3b), maturation (synaptophysin) and neurite growth (β-III-tubulin) after an additional 15 days. We further examined the physiologic differentiation of these hESC-derived RGC-like progeny to those differentiated in vitro from primary rodent retinal progenitor cells (RPCs) with calcium imaging, and found that both populations demonstrate the immature RGC-like response to muscimol, a GABAA receptor agonist. By one week after transplant to the adult rat eye by intravitreal injection, the human RGC-like cells successfully migrated into the ganglion cell layer.Our protocol provides a novel, short, and cost-effective approach for RGC differentiation from hESCs, and may broaden the scope for cell replacement therapy in RGC-related optic neuropathies such as glaucoma.
View details for DOI 10.3233/RNN-190941
View details for PubMedID 31815704
SALT Trial: Steroids after Laser Trabeculoplasty: Impact of Short-Term Anti-inflammatory Treatment on Selective Laser Trabeculoplasty Efficacy.
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).Double-masked, randomized, placebo-controlled, dual-center, multisurgeon trial.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.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.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.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).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.
View details for DOI 10.1016/j.ophtha.2019.05.032
View details for PubMedID 31444008
Regulation of Neuronal Survival and Axon Growth by a Perinuclear cAMP Compartment.
The Journal of neuroscience : the official journal of the Society for Neuroscience
Cyclic-AMP (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 (PKA) 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 STATEMENTcAMP 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 due to 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.
View details for PubMedID 31097623
MTP18 is a Novel Regulator of Mitochondrial Fission in CNS Neuron Development, Axonal Growth, and Injury Responses.
2019; 9 (1): 10669
The process of mitochondrial fission-fusion has been implicated in diverse neuronal roles including neuronal survival, axon degeneration, and axon regeneration. However, whether increased fission or fusion is beneficial for neuronal health and/or axonal growth is not entirely clear, and is likely situational and cell type-dependent. In searching for mitochondrial fission-fusion regulating proteins for improving axonal growth within the visual system, we uncover that mitochondrial fission process 1,18 kDa (MTP18/MTFP1), a pro-fission protein within the CNS, is critical to maintaining mitochondrial size and volume under normal and injury conditions, in retinal ganglion cells (RGCs). We demonstrate that MTP18's expression is regulated by transcription factors involved in axonal growth, Kruppel-like factor (KLF) transcription factors-7 and -9, and that knockdown of MTP18 promotes axon growth. This investigation exposes MTP18's previously unexplored role in regulating mitochondrial fission, implicates MTP18 as a downstream component of axon regenerative signaling, and ultimately lays the groundwork for investigations on the therapeutic efficacy of MTP18 expression suppression during CNS axon degenerative events.
View details for DOI 10.1038/s41598-019-46956-5
View details for PubMedID 31337818
Magnetic Human Corneal Endothelial Cell Transplant: Delivery, Retention, and Short-Term Efficacy.
Investigative ophthalmology & visual science
2019; 60 (7): 2438–48
Corneal endothelial dysfunction leads to corneal edema, pain, and vision loss. Adequate animal models are needed to study the safety and efficacy of novel cell therapies as an alternative to corneal transplantation.Primary human corneal endothelial cells (HCECs) were isolated from cadaveric donor corneas, expanded in vitro, transduced to express green fluorescent protein (GFP), loaded with superparamagnetic nanoparticles, and injected into the anterior chamber of adult rabbits immediately after endothelial cell or Descemet's membrane stripping. The same volume of balanced salt solution plus (BSS+) was injected in control eyes. We compared different models for inducing corneal edema in rabbits, and examined the ability of transplanted HCECs to reduce corneal edema over time by measuring central corneal thickness and tracking corneal clarity. GFP-positive donor cells were tracked in vivo using optical coherence tomography (OCT) fluorescence angiography module, and the transplanted cells were confirmed by human nuclei immunostaining.Magnetic HCECs integrated onto the recipient corneas with intact Descemet's membrane, and donor identity was confirmed by GFP expression and immunostaining for human nuclei marker. Donor HCECs formed a monolayer on the posterior corneal surface and expressed HCEC functional markers of tight junction formation. No GFP-positive cells were observed in the trabecular meshwork or on the iris, and intraocular pressure remained stable through the length of the study.Our results demonstrate magnetic cell-based therapy efficiently delivers HCECs to restore corneal transparency without detectable toxicity or adverse effect on intraocular pressure. Magnetic delivery of HCECs may enhance corneal function and should be explored further for human therapies.
View details for DOI 10.1167/iovs.18-26001
View details for PubMedID 31158276
Clinical Grade Magnetic Human Corneal Endothelial Cells for Cell Therapy
ASSOC RESEARCH VISION OPHTHALMOLOGY INC. 2018
View details for Web of Science ID 000442912506247
Dynamics of human ON and OFF Visual Pathways
ASSOC RESEARCH VISION OPHTHALMOLOGY INC. 2018
View details for Web of Science ID 000442932805075
Development of a Porcine Optic Nerve Injury Model
ASSOC RESEARCH VISION OPHTHALMOLOGY INC. 2018
View details for Web of Science ID 000442912500275
Generation and purification of functional corneal endothelium-like cells differentiated from human embryonic stem cells
ASSOC RESEARCH VISION OPHTHALMOLOGY INC. 2018
View details for Web of Science ID 000442912506248
A Cell Culture Approach to Optimized Human Corneal Endothelial Cell Function.
Investigative ophthalmology & visual science
2018; 59 (3): 1617–29
Cell-based therapies to replace corneal endothelium depend on culture methods to optimize human corneal endothelial cell (HCEC) function and minimize endothelial-mesenchymal transition (EnMT). Here we explore contribution of low-mitogenic media on stabilization of phenotypes in vitro that mimic those of HCECs in vivo.HCECs were isolated from cadaveric donor corneas and expanded in vitro, comparing continuous presence of exogenous growth factors ("proliferative media") to media without those factors ("stabilizing media"). Identity based on canonical morphology and expression of surface marker CD56, and function based on formation of tight junction barriers measured by trans-endothelial electrical resistance assays (TEER) were assessed.Primary HCECs cultured in proliferative media underwent EnMT after three to four passages, becoming increasingly fibroblastic. Stabilizing the cells before each passage by switching them to a media low in mitogenic growth factors and serum preserved canonical morphology and yielded a higher number of cells. HCECs cultured in stabilizing media increased both expression of the identity marker CD56 and also tight junction monolayer integrity compared to cells cultured without stabilization.HCECs isolated from donor corneas and expanded in vitro with a low-mitogenic media stabilizing step before each passage demonstrate more canonical structural and functional features and defer EnMT, increasing the number of passages and total canonical cell yield. This approach may facilitate development of HCEC-based cell therapies.
View details for PubMedID 29625488
Vision Loss after Intravitreal Injection of Autologous "Stem Cells" for AMD
NEW ENGLAND JOURNAL OF MEDICINE
2017; 376 (11): 1047-1053
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.
View details for DOI 10.1056/NEJMoa1609583
View details for Web of Science ID 000396403700009
View details for PubMedID 28296617
Soluble Adenylyl Cyclase Is Required for Retinal Ganglion Cell and Photoreceptor Differentiation.
Investigative ophthalmology & visual science
2016; 57 (11): 5083-5092
We have previously demonstrated that soluble adenylyl cyclase (sAC) is necessary for retinal ganglion cell (RGC) survival and axon growth. Here, we further investigate the role of sAC in neuronal differentiation during retinal development.Chx10 or Math5 promoter-driven Cre-Lox recombination were used to conditionally delete sAC from early and intermediate retinal progenitor cells during retinal development. We examined cell type-specific markers expressed by retinal cells to estimate their relative numbers and characterize retinal laminar morphology by immunofluorescence in adult and newborn mice.Retinal ganglion cell and amacrine cell markers were significantly lower in the retinas of adult Math5cre/sACfl/fl and Chx10cre/sACfl/fl mice than in those of wild-type controls. The effect on RGC development was detectable as early as postnatal day 1 and deleting sAC in either Math5- or Chx10-expressing retinal progenitor cells also reduced nerve fiber layer thickness into adulthood. The thickness of the photoreceptor layer was slightly but statistically significantly decreased in both the newborn Chx10cre/sACfl/fl and Math5cre/sACfl/fl mice, but this reduction and abnormal morphology persisted in the adults in only the Chx10cre/sACfl/fl mice.sAC plays an important role in the early retinal development of RGCs as well as in the development of amacrine cells and to a lesser degree photoreceptors.
View details for DOI 10.1167/iovs.16-19465
View details for PubMedID 27679853
View details for PubMedCentralID PMC5053116
- Cell types differ in global coordination of splicing and proportion of highly expressed genes SCIENTIFIC REPORTS 2016; 6
Cell types differ in global coordination of splicing and proportion of highly expressed genes.
2016; 6: 32249-?
Balance in the transcriptome is regulated by coordinated synthesis and degradation of RNA molecules. Here we investigated whether mammalian cell types intrinsically differ in global coordination of gene splicing and expression levels. We analyzed RNA-seq transcriptome profiles of 8 different purified mouse cell types. We found that different cell types vary in proportion of highly expressed genes and the number of alternatively spliced transcripts expressed per gene, and that the cell types that express more variants of alternatively spliced transcripts per gene are those that have higher proportion of highly expressed genes. Cell types segregated into two clusters based on high or low proportion of highly expressed genes. Biological functions involved in negative regulation of gene expression were enriched in the group of cell types with low proportion of highly expressed genes, and biological functions involved in regulation of transcription and RNA splicing were enriched in the group of cell types with high proportion of highly expressed genes. Our findings show that cell types differ in proportion of highly expressed genes and the number of alternatively spliced transcripts expressed per gene, which represent distinct properties of the transcriptome and may reflect intrinsic differences in global coordination of synthesis, splicing, and degradation of RNA molecules.
View details for DOI 10.1038/srep32249
View details for PubMedID 27577089
Novel Roles and Mechanism for Krüppel-like Factor 16 (KLF16) Regulation of Neurite Outgrowth and Ephrin Receptor A5 (EphA5) Expression in Retinal Ganglion Cells.
journal of biological chemistry
2016; 291 (35): 18084-18095
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.
View details for DOI 10.1074/jbc.M116.732339
View details for PubMedID 27402841
View details for PubMedCentralID PMC5000058
Topical administration of a Rock/Net inhibitor promotes retinal ganglion cell survival and axon regeneration after optic nerve injury.
Experimental eye research
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.
View details for DOI 10.1016/j.exer.2016.07.006
View details for PubMedID 27443501
- NEUROREGENERATION. Promoting CNS repair. Science 2016; 353 (6294): 30-31
Novel Identity and Functional Markers for Human Corneal Endothelial Cells
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE
2016; 57 (6): 2749-2762
Human corneal endothelial cell (HCEC) density decreases with age, surgical complications, or disease, leading to vision impairment. Such endothelial dysfunction is an indication for corneal transplantation, although there is a worldwide shortage of transplant-grade tissue. To overcome the current poor donor availability, here we isolate, expand, and characterize HCECs in vitro as a step toward cell therapy.Human corneal endothelial cells were isolated from cadaveric corneas and expanded in vitro. Cell identity was evaluated based on morphology and immunocytochemistry, and gene expression analysis and flow cytometry were used to identify novel HCEC-specific markers. The functional ability of HCEC to form barriers was assessed by transendothelial electrical resistance (TEER) assays.Cultured HCECs demonstrated canonical morphology for up to four passages and later underwent endothelial-to-mesenchymal transition (EnMT). Quality of donor tissue influenced cell measures in culture including proliferation rate. Cultured HCECs expressed identity markers, and microarray analysis revealed novel endothelial-specific markers that were validated by flow cytometry. Finally, canonical HCECs expressed higher levels of CD56, which correlated with higher TEER than fibroblastic HCECs.In vitro expansion of HCECs from cadaveric donor corneas yields functional cells identifiable by morphology and a panel of novel markers. Markers described correlated with function in culture, suggesting a basis for cell therapy for corneal endothelial dysfunction.
View details for DOI 10.1167/iovs.15-18826
View details for Web of Science ID 000378041700044
View details for PubMedID 27196322
View details for PubMedCentralID PMC4884060
Ocular Stem Cell Research from Basic Science to Clinical Application: A Report from Zhongshan Ophthalmic Center Ocular Stem Cell Symposium
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
2016; 17 (3)
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.
View details for DOI 10.3390/ijms17030415
View details for Web of Science ID 000373712800084
View details for PubMedID 27102165
View details for PubMedCentralID PMC4813266
The Frequency of Optical Coherence Tomography Testing in Glaucoma at a Single Academic Medical Center
JOURNAL OF GLAUCOMA
2016; 25 (3): E241-E247
To determine the frequency of optical coherence tomography (OCT) examinations compared with clinical examinations and visual field (VF) tests in patients with 5 types of glaucoma.A retrospective, longitudinal cohort study was conducted of 5154 patients treated between 2003 and 2010 at a single academic medical center. Patients were classified using billing records as having primary open-angle glaucoma, low-tension open-angle glaucoma (NTG), pigmentary open-angle glaucoma, chronic angle-closure glaucoma, or pseudoexfoliation glaucoma. Analysis of variance, χ test, and exact χ test were performed to identify associations between glaucoma type and test frequency.Pigmentary open-angle glaucoma and NTG patients had a higher rate of undergoing at least 2 VFs (94.4%, 94.9%), and chronic angle-closure glaucoma patients had a lower rate of undergoing at least 2 OCTs (25.3%) than all other glaucoma types. NTG patients also had the highest rate of undergoing at least 2 OCTs and at least 2 VFs (36.6%). Overall, the rate of clinical examinations (2.68 examinations/y) exceeded the rates of OCTs (1.39 examinations/y), which exceeded the rate of VF tests (1.24 tests/y). There were no differences in OCT frequency between glaucoma types (0.91 to 1.63 OCTs/y). Within each glaucoma diagnosis, patients had clinical examinations more frequently than OCTs and clinical examinations more frequently than VFs. Primary open-angle glaucoma and pseudoexfoliation glaucoma patients also had OCTs more frequently than VFs. More patients had at least 2 VF tests than at least 2 OCTs (4481 vs. 1679).The relative use of clinical examinations, VF testing, and OCT imaging varies among glaucoma diagnoses.
View details for DOI 10.1097/IJG.0000000000000306
View details for Web of Science ID 000374821000020
View details for PubMedID 26372155
View details for PubMedCentralID PMC4885912
Report on the National Eye Institute Audacious Goals Initiative: Regenerating the Optic Nerve.
Investigative ophthalmology & visual science
2016; 57 (3): 1271-1275
The National Eye Institute (NEI) hosted a workshop on November 19, 2014, as part of the Audacious Goals Initiative (AGI), an NEI-led effort to rapidly expand therapies for eye diseases through coordinated research funding. The central audacious goal aims to demonstrate by 2025 the restoration of usable vision in humans through the regeneration of neurons and neural connections in the eye and visual system. This workshop focused on identifying promising strategies for optic nerve regeneration. Its principal objective was to solicit input on future AGI-related funding announcements, and specifically to ask, where are we now in our scientific progress, and what progress should we reach for in the coming years? A full report was generated as a white paper posted on the NEI Web site; this report summarizes the discussion and outcomes from the meeting and serves as guidance for future funding of research that focuses on optic nerve regeneration.
View details for DOI 10.1167/iovs.15-18500
View details for PubMedID 26990163
Transplanted neurons integrate into adult retinas and respond to light.
2016; 7: 10472-?
Retinal ganglion cells (RGCs) degenerate in diseases like glaucoma and are not replaced in adult mammals. Here we investigate whether transplanted RGCs can integrate into the mature retina. We have transplanted GFP-labelled RGCs into uninjured rat retinas in vivo by intravitreal injection. Transplanted RGCs acquire the general morphology of endogenous RGCs, with axons orienting towards the optic nerve head of the host retina and dendrites growing into the inner plexiform layer. Preliminary data show in some cases GFP(+) axons extending within the host optic nerves and optic tract, reaching usual synaptic targets in the brain, including the lateral geniculate nucleus and superior colliculus. Electrophysiological recordings from transplanted RGCs demonstrate the cells' electrical excitability and light responses similar to host ON, ON-OFF and OFF RGCs, although less rapid and with greater adaptation. These data present a promising approach to develop cell replacement strategies in diseased retinas with degenerating RGCs.
View details for DOI 10.1038/ncomms10472
View details for PubMedID 26843334
Control of Retinal Ganglion Cell Positioning and Neurite Growth: Combining 3D Printing with Radial Electrospun Scaffolds
TISSUE ENGINEERING PART A
2016; 22 (3-4): 286-294
Retinal ganglion cells (RGCs) are responsible for the transfer of signals from the retina to the brain. As part of the central nervous system, RGCs are unable to regenerate following injury, and implanted cells have limited capacity to orient and integrate in vivo. During development, secreted guidance molecules along with signals from extracellular matrix and the vasculature guide cell positioning, for example, around the fovea, and axon outgrowth; however, these changes are temporally regulated and are not the same in the adult. Here, we combine electrospun cell transplantation scaffolds capable of RGC neurite guidance with thermal inkjet 3D cell printing techniques capable of precise positioning of RGCs on the scaffold surface. Optimal printing parameters are developed for viability, electrophysiological function and, neurite pathfinding. Different media, commonly used to promote RGC survival and growth, were tested under varying conditions. When printed in growth media containing both brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF), RGCs maintained survival and normal electrophysiological function, and displayed radial axon outgrowth when printed onto electrospun scaffolds. These results demonstrate that 3D printing technology may be combined with complex electrospun surfaces in the design of future retinal models or therapies.
View details for DOI 10.1089/ten.tea.2015.0373
View details for Web of Science ID 000369987900011
View details for PubMedID 26729061
View details for PubMedCentralID PMC4779292
Krüppel-Like Factor 4 (KLF4) Is Not Required for Retinal Cell Differentiation.
2016; 3 (1)
During early vertebrate eye development, a regulatory network of transcription factors regulates retinal cell differentiation and survival into adulthood. Among those factors, Krüppel-like factor 4 (KLF4) plays the dual role of maintaining the stem cell status of retinal progenitors cells and repressing the intrinsic axon regeneration ability in retinal ganglion cells (RGCs) after injury. This study further investigated whether KLF4 plays a role in early retinal cell differentiation or survival into adulthood. We examined different types of retinal neurons, including RGCs, amacrine cells, bipolar cells, Müller cells, and photoreceptor cells, in adult mice in which KLF4 was conditionally deleted in early retinal development using Chx10-promoted Cre by immunohistochemistry. We compared the numbers of retinal neurons and the thickness of photoreceptor and nerve fiber layers between Chx10-Cre-driven KLF4 deletion mice and wild-type mice. There was no significant difference in cell number among any of the retinal cell types or in photoreceptor layer thickness with KLF4 deletion during early development. The thickness of axon bundles in the nerve fiber layer in the Chx10 conditional KLF4 knock-out mice was greater than that in wild-type mice. These results suggest that KLF4 is not required for retinal cell differentiation or survival, but does normally limit retinal ganglion cell axon bundle thickness. These data support a hypothesis that KLF4 suppresses axon growth during development.
View details for DOI 10.1523/ENEURO.0117-15.2016
View details for PubMedID 27022622
View details for PubMedCentralID PMC4770008
Is uveitis associated with topiramate use? A cumulative review.
Clinical ophthalmology (Auckland, N.Z.)
2016; 10: 1467-1470
Occasional reports of uveitis following topiramate use necessitated an investigation of relevant cases from safety databases and published biomedical literature. Data mining of the Food and Drug Administration Adverse Event Reporting System and cumulative review of cases from the global safety database (sponsor database) and published literature were conducted to assess association between topiramate use and uveitis. The Food and Drug Administration Adverse Event Reporting System search identified disproportional reporting of uveitis (n=23) and related terms (choroidal detachment, n=25; iridocyclitis, n=17). The postmarketing reporting frequency of uveitis and related events from the global safety database and based on an estimated topiramate exposure of 11,185,740 person-years from launch to April 2015 was 0.38 per 100,000 person-years and assigned as very rare. A total of 14 potential uveitis cases were identified from the cumulative review. Seven of these 14 cases were complicated by inadequate documentation, appearance of uveitic signs following drug withdrawal, or concurrent use of other sulfonamides. In acute angle-closure glaucoma and uveal effusions cases, insufficient evidence for underlying inflammation suggested that uveitis was not a component. Only seven of 14 cases were well documented, potentially topiramate-associated uveitis cases. Uveitis may occur in the setting of topiramate use only in very rare instances. Current evidence did not reveal a dose- or duration-dependent relationship between uveitis and topiramate use.
View details for DOI 10.2147/OPTH.S104847
View details for PubMedID 27536060
- Efficient Generation of Human Embryonic Stem Cell-Derived Corneal Endothelial Cells by Directed Differentiation PLOS ONE 2015; 10 (12)
Muscle A-Kinase Anchoring Protein-a is an Injury-Specific Signaling Scaffold Required for Neurotrophic- and Cyclic Adenosine Monophosphate-Mediated Survival.
2015; 2 (12): 1880-1887
Neurotrophic factor and cAMP-dependent signaling promote the survival and neurite outgrowth of retinal ganglion cells (RGCs) after injury. However, the mechanisms conferring neuroprotection and neuroregeneration downstream to these signals are unclear. We now reveal that the scaffold protein muscle A-kinase anchoring protein-α (mAKAPα) is required for the survival and axon growth of cultured primary RGCs. Although genetic deletion of mAKAPα early in prenatal RGC development did not affect RGC survival into adulthood, nor promoted the death of RGCs in the uninjured adult retina, loss of mAKAPα in the adult increased RGC death after optic nerve crush. Importantly, mAKAPα was required for the neuroprotective effects of brain-derived neurotrophic factor and cyclic adenosine-monophosphate (cAMP) after injury. These results identify mAKAPα as a scaffold for signaling in the stressed neuron that is required for RGC neuroprotection after optic nerve injury.
View details for DOI 10.1016/j.ebiom.2015.10.025
View details for PubMedID 26844267
- Rat Model of Photochemically-Induced Posterior Ischemic Optic Neuropathy JOVE-JOURNAL OF VISUALIZED EXPERIMENTS 2015
Treatment of Inherited Eye Defects by Systemic Hematopoietic Stem Cell Transplantation
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE
2015; 56 (12): 7214-7223
Cystinosis is caused by a deficiency in the lysosomal cystine transporter, cystinosin (CTNS gene), resulting in cystine crystal accumulation in tissues. In eyes, crystals accumulate in the cornea causing photophobia and eventually blindness. Hematopoietic stem progenitor cells (HSPCs) rescue the kidney in a mouse model of cystinosis. We investigated the potential for HSPC transplantation to treat corneal defects in cystinosis.We isolated HSPCs from transgenic DsRed mice and systemically transplanted irradiated Ctns-/- mice. A year posttransplantation, we investigated the fate and function of HSPCs by in vivo confocal and fluorescence microscopy (IVCM), quantitative RT-PCR (RT-qPCR), mass spectrometry, histology, and by measuring the IOP. To determine the mechanism by which HSPCs may rescue disease cells, we transplanted Ctns-/- mice with Ctns-/- DsRed HSPCs virally transduced to express functional CTNS-eGFP fusion protein.We found that a single systemic transplantation of wild-type HSPCs prevented ocular pathology in the Ctns-/- mice. Engraftment-derived HSPCs were detected within the cornea, and also in the sclera, ciliary body, retina, choroid, and lens. Transplantation of HSPC led to substantial decreases in corneal cystine crystals, restoration of normal corneal thickness, and lowered IOP in mice with high levels of donor-derived cell engraftment. Finally, we found that HSPC-derived progeny differentiated into macrophages, which displayed tunneling nanotubes capable of transferring cystinosin-bearing lysosomes to diseased cells.To our knowledge, this is the first demonstration that HSPCs can rescue hereditary corneal defects, and supports a new potential therapeutic strategy for treating ocular pathologies.
View details for DOI 10.1167/iovs.15-17107
View details for Web of Science ID 000368238200027
View details for PubMedCentralID PMC4640476
Mapping the 3D Connectivity of the Rat Inner Retinal Vascular Network Using OCT Angiography
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE
2015; 56 (10): 5785-5793
The purpose of this study is to demonstrate three-dimensional (3D) graphing based on optical coherence tomography (OCT) angiography for characterization of the inner retinal vascular architecture and determination of its topologic principles.Rat eyes (N = 3) were imaged with a 1300-nm spectral/Fourier domain OCT microscope. A topologic model of the inner retinal vascular network was obtained from OCT angiography data using a combination of automated and manually-guided image processing techniques. Using a resistive network model, with experimentally-quantified flow in major retinal vessels near the optic nerve head as boundary conditions, theoretical changes in the distribution of flow induced by vessel dilations were inferred.A topologically-representative 3D vectorized graph of the inner retinal vasculature, derived from OCT angiography data, is presented. The laminar and compartmental connectivity of the vasculature are characterized. In contrast to sparse connectivity between the superficial vitreal vasculature and capillary plexuses of the inner retina, connectivity between the two capillary plexus layers is dense. Simulated dilation of single arterioles is shown to produce both localized and lamina-specific changes in blood flow, while dilation of capillaries in a given retinal vascular layer is shown to lead to increased total flow in that layer.Our graphing and modeling data suggest that vascular architecture enables both local and lamina-specific control of blood flow in the inner retina. The imaging, graph analysis, and modeling approach presented here will help provide a detailed characterization of vascular changes in a variety of retinal diseases, both in experimental preclinical models and human subjects.
View details for DOI 10.1167/iovs.15-17210
View details for Web of Science ID 000368426300011
View details for PubMedID 26325417
View details for PubMedCentralID PMC4559217
In vivo imaging of axonal transport of mitochondria in the diseased and aged mammalian CNS
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2015; 112 (33): 10515-10520
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.
View details for DOI 10.1073/pnas.1509879112
View details for Web of Science ID 000359738300093
View details for PubMedID 26240337
View details for PubMedCentralID PMC4547257
The N-terminal Set-beta Protein Isoform Induces Neuronal Death
JOURNAL OF BIOLOGICAL CHEMISTRY
2015; 290 (21): 13417-13426
Set-β protein plays different roles in neurons, but the diversity of Set-β neuronal isoforms and their functions have not been characterized. The expression and subcellular localization of Set-β are altered in Alzheimer disease, cleavage of Set-β leads to neuronal death after stroke, and the full-length Set-β 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-β 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-β-specific exon were the most highly expressed isoforms. We also identified a novel, alternatively spliced Set-β transcript lacking the nuclear localization signal and demonstrated that the full-length (∼39-kDa) Set-β is localized predominantly in the nucleus, whereas a shorter (∼25-kDa) Set-β isoform is localized predominantly in the cytoplasm. Finally, we show that an N-terminal Set-β cleavage product can induce neuronal death.
View details for DOI 10.1074/jbc.M114.633883
View details for Web of Science ID 000354975700037
View details for PubMedID 25833944
View details for PubMedCentralID PMC4505589
Promoting filopodial elongation in neurons by membrane-bound magnetic nanoparticles
NANOMEDICINE-NANOTECHNOLOGY BIOLOGY AND MEDICINE
2015; 11 (3): 559-567
Filopodia are 5-10 μm long processes that elongate by actin polymerization, and promote axon growth and guidance by exerting mechanical tension and by molecular signaling. Although axons elongate in response to mechanical tension, the structural and functional effects of tension specifically applied to growth cone filopodia are unknown. Here we developed a strategy to apply tension specifically to retinal ganglion cell (RGC) growth cone filopodia through surface-functionalized, membrane-targeted superparamagnetic iron oxide nanoparticles (SPIONs). When magnetic fields were applied to surface-bound SPIONs, RGC filopodia elongated directionally, contained polymerized actin filaments, and generated retrograde forces, behaving as bona fide filopodia. Data presented here support the premise that mechanical tension induces filopodia growth but counter the hypothesis that filopodial tension directly promotes growth cone advance. Future applications of these approaches may be used to induce sustained forces on multiple filopodia or other subcellular microstructures to study axon growth or cell migration. From the clinical editor: Mechanical tension to the tip of filopodia is known to promote axonal growth. In this article, the authors used superparamagnetic iron oxide nanoparticles (SPIONs) targeted specifically to membrane molecules, then applied external magnetic field to elicit filopodial elongation, which provided a tool to study the role of mechanical forces in filopodia dynamics and function.
View details for DOI 10.1016/j.nano.2014.11.011
View details for Web of Science ID 000352081100007
View details for PubMedID 25596077
View details for PubMedCentralID PMC4691347
Magnetic field-guided cell delivery with nanoparticle-loaded human corneal endothelial cells
NANOMEDICINE-NANOTECHNOLOGY BIOLOGY AND MEDICINE
2015; 11 (3): 499-509
To improve the delivery and integration of cell therapy using magnetic cell guidance for replacement of corneal endothelium, here we assess magnetic nanoparticles' (MNPs') effects on human corneal endothelial cells (HCECs) in vitro. Biocompatible, 50 nm superparamagnetic nanoparticles endocytosed by cultured HCECs induced no short- or long-term change in viability or identity. Assessment of guidance of the magnetic HCECs in the presence of different magnet shapes and field strengths showed a 2.4-fold increase in delivered cell density compared to gravity alone. After cell delivery, HCECs formed a functional monolayer, with no difference in tight junction formation between MNP-loaded and control HCECs. These data suggest that nanoparticle-mediated magnetic cell delivery may increase the efficiency of cell delivery without compromising HCEC survival, identity or function. Future studies may assess the safety and efficacy of this therapeutic modality in vivo. From the clinical editor: The authors show in this article that magnetic force facilitates the delivery of human corneal endothelial cells loaded by superparamagnetic nanoparticles to cornea, without changing their morphology, identity or functional properties. This novel idea can potentially have vast impact in the treatment of corneal endothelial dystrophies by providing self-endothelial cells after ex-vivo expansion.
View details for DOI 10.1016/j.nano.2014.12.002
View details for Web of Science ID 000352081100002
View details for PubMedID 25596075
View details for PubMedCentralID PMC4691344
Clinical and Electrophysiologic Characteristics of a Large Kindred with X-Linked Retinitis Pigmentosa Associated with the RPGR Locus
2015; 36 (4): 321-326
To phenotypically and genotypically characterize a large Puerto Rican kindred with X-linked retinitis pigmentosa associated with a novel RP GTPase regulator (RPGR) genotype.A total of 100 family members of a single kindred with X-linked RP were evaluated with ophthalmic examinations and blood DNA analysis. Visual fields, OCT, and full-field ERG were obtained on all affected males and carriers.Of the 100 family members examined, 13 were affected males and 18 were carriers. A deletion of 2 base pair of the RPGR gene in the ORF15 region at position c.2267-2268 (Lys756del2aaAG hemi) was identified with the affected and carriers. Best eye visual acuity was correlated with age (Spearman coefficient = 0.95) with hand-motion acuity by age 35 and light perception to no light perception by age 50-60. Visual fields were minimally plottable by age 40, and ERG responses reached non-detectable levels by late teens. Carriers had no or mild visual symptoms. All carriers had visual acuity of at least 20/50 or better in one eye, and the amount of retinal degeneration was variable with ERG responses ranging from severely impaired to normal.Profound visual loss occurred by the second decade of life with progression to near no light perception by age 60 in this kindred of X-linked RP associated with the RPGR genotype. Female carriers maintained visual acuity with age and were identifiable by clinical and ERG examination. The information from this study is important to determine the optimal age for intervention, as new RP treatments are being developed and tested.
View details for DOI 10.3109/13816810.2014.886267
View details for Web of Science ID 000369858800006
View details for PubMedID 24555744
Prevalence of comorbid retinal disease in patients with glaucoma at an academic medical center.
Clinical ophthalmology (Auckland, N.Z.)
2015; 9: 1275-1284
Patients with various retinal diseases and patients who have undergone retinal procedures and surgeries have an increased risk of developing ocular hypertension and glaucoma. Little is known about the epidemiology of comorbid retinal diseases in glaucoma patients. This study evaluated the prevalence of retinal comorbidities in a population of patients with five types of glaucoma.A longitudinal, retrospective study was conducted using International Classification of Disease (ICD-9) billing records from 2003 to 2010 at an academic medical center. Patients were classified as having primary open-angle glaucoma (POAG), low tension open-angle glaucoma (NTG), pigmentary open-angle glaucoma, chronic-angle closure glaucoma (CACG), or pseudoexfoliation glaucoma (PXG) if they had at least three clinic visits with the same ICD-9 code. Patients were classified as having a retinal comorbidity if they had two visits with the same code. Variables were analyzed with the independent t-test, χ (2) test, analysis of variance, or Fisher's exact test.A total of 5,154 patients had glaucoma, and 14.8% of these had a retinal comorbidity. The prevalence of comorbid retinal disease was higher in patients with POAG (15.7%) than in those with NTG (10.7%), PXG (10.1%), or pigmentary open-angle glaucoma (3.7%; P<0.05). Two hundred and two patients had diabetic retinopathy, with POAG patients (4.5%) having a higher prevalence than those with CACG (1.4%) or PXG (0.6%; P<0.001). There were 297 patients who had macular degeneration and both POAG (2.0%) and PXG patients (2.9%) had a higher prevalence of nonexudative macular degeneration than those with CACG (0%; P<0.01). Patients with comorbid retinal disease had a higher prevalence of blindness and low vision than those without comorbid retinal disease (1.97% versus 1.02%, P=0.02).The high prevalence of comorbid retinal disease and the nearly twofold increase in blindness and low vision in this population demonstrate the need for ophthalmologists to determine if patients have multiple etiologies for their vision loss. The higher prevalence of certain retinal diseases in POAG patients may reflect common pathophysiological processes that warrant further investigation.
View details for DOI 10.2147/OPTH.S85851
View details for PubMedID 26203217
View details for PubMedCentralID PMC4508087
Retinal ganglion cell polarization using immobilized guidance cues on a tissue-engineered scaffold
2014; 10 (12): 4939-4946
Cell transplantation therapies to treat diseases related to dysfunction of retinal ganglion cells (RGCs) are limited in part by an inability to navigate to the optic nerve head within the retina. During development, RGCs are guided by a series of neurotrophic factors and guidance cues; however, these factors and their receptors on the RGCs are developmentally regulated and often not expressed during adulthood. Netrin-1 is a guidance factor capable of guiding RGCs in culture and relevant to guiding RGC axons toward the optic nerve head in vivo. Here we immobilized Netrin-1 using UV-initiated crosslinking to form a gradient capable of guiding the axonal growth of RGCs on a radial electrospun scaffold. Netrin-gradient scaffolds promoted both the percentage of RGCs polarized with a single axon, and also the percentage of cells polarized toward the scaffold center, from 31% to 52%. Thus, an immobilized protein gradient on a radial electrospun scaffold increases RGC axon growth in a direction consistent with developmental optic nerve head guidance, and may prove beneficial for use in cell transplant therapies for the treatment of glaucoma and other optic neuropathies.
View details for DOI 10.1016/j.actbio.2014.08.032
View details for Web of Science ID 000345468300002
View details for PubMedID 25194930
View details for PubMedCentralID PMC4254021
The role of soluble adenylyl cyclase in neurite outgrowth
BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR BASIS OF DISEASE
2014; 1842 (12): 2561-2568
Axon regeneration in the mature central nervous system is limited by extrinsic inhibitory signals and a postnatal decline in neurons' intrinsic growth capacity. Neuronal levels of the second messenger cAMP are important in regulating both intrinsic growth capacity and neurons' responses to extrinsic factors. Approaches which increase intracellular cAMP in neurons enhance neurite outgrowth and facilitate regeneration after injury. Thus, understanding the factors which affect cAMP in neurons is of potential therapeutic importance. Recently, soluble adenylyl cyclase (sAC, ADCY10), the ubiquitous, non-transmembrane adenylyl cyclase, was found to play a key role in neuronal survival and axon growth. sAC is activated by bicarbonate and cations and may translate physiologic signals from metabolism and electrical activity into a neuron's decision to survive or regenerate. Here we critically review the literature surrounding sAC and cAMP signaling in neurons to further elucidate the potential role of sAC signaling in neurite outgrowth and regeneration. This article is part of a Special Issue entitled: The role of soluble adenylyl cyclase in health and disease.
View details for DOI 10.1016/j.bbadis.2014.07.012
View details for Web of Science ID 000347590800005
View details for PubMedID 25064589
View details for PubMedCentralID PMC4262618
Regenerative Cell Therapy for Corneal Endothelium.
Current ophthalmology reports
2014; 2 (3): 81-90
Endothelial cell dysfunction as in Fuchs dystrophy or pseudophakic bullous keratopathy, and the limited regenerative capacity of human corneal endothelial cells (HCECs), drive the need for corneal transplant. In response to limited donor corneal availability, significant effort has been directed towards cell therapy as an alternative to surgery. Stimulation of endogenous progenitors, or transplant of stem cell-derived HCECs or in vitro-expanded, donor-derived HCECs could replace traditional surgery with regenerative therapy. Ex vivo expansion of HCECs is technically challenging, and the basis for molecular identification of functional HCECs is not established. Delivery of cells to the inner layer of the human cornea is another challenge: different techniques, from simple injection to artificial corneal scaffolds, are being investigated. Despite remaining questions, corneal endothelial cell therapies, translated to the clinic, represent the future for the treatment of corneal endotheliopathies.
View details for PubMedID 25328857
Molecular mechanisms of the suppression of axon regeneration by KLF transcription factors
NEURAL REGENERATION RESEARCH
2014; 9 (15): 1418-1421
Molecular mechanisms of the Krüppel-like family of transcription factors (KLFs) have been studied more in proliferating cells than in post-mitotic cells such as neurons. We recently found that KLFs regulate intrinsic axon growth ability in central nervous system (CNS) neurons including retinal ganglion cells, and hippocampal and cortical neurons. With at least 15 of 17 KLF family members expressed in neurons and at least 5 structurally unique subfamilies, it is important to determine how this complex family functions in neurons to regulate the intricate genetic programs of axon growth and regeneration. By characterizing the molecular mechanisms of the KLF family in the nervous system, including binding partners and gene targets, and comparing them to defined mechanisms defined outside the nervous system, we may better understand how KLFs regulate neurite growth and axon regeneration.
View details for DOI 10.4103/1673-5374.139454
View details for Web of Science ID 000342650400002
View details for PubMedID 25317150
View details for PubMedCentralID PMC4192940
Isolation and Characterization of Mesenchymal Progenitor Cells From Human Orbital Adipose Tissue
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE
2014; 55 (8): 4842-4852
Adipose-derived stem cells (ASCs) have gained importance due to their myriad potential clinical applications. We hypothesize that progenitor cells also exist besides those conventionally isolated from the stromal vascular fraction (SVF).Central and medial orbital adipose tissues obtained from patients during eyelid surgery were digested with collagenase for 3 or 16 hours at 37°C with or without shaking. After centrifugation, the remaining cell pellet was resuspended and filtered to yield flow through in SVF and retained cells (RC) on the filter. Single cells from RC and SVF were cultured on 5% coated Matrigel in serum-free modified embryonic stem cells medium (MESCM) for 10 passages. The progenitor status was evaluated by the expression of a number of markers by qPCR and immunofluorescence staining as well as their plasticity for endothelial and tri-lineage differentiation.Type I collagenase digestion for 3 hours under shaking was significantly less effective in releasing progenitor cells than collagenase A digestion for 16 hours without shaking. Following filtration, cells in SVF and RC, of which the latter were tangled in collagen IV-containing matrix, expressed different markers of progenitor cells. Cells from SVF and RC could be expanded for 10 passages on coated Matrigel in MESCM and exhibited similar or better potential to differentiate into vascular endothelial cells, chondrocytes, osteocytes, and adipocytes than SVF cells expanded on plastic in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS).Different progenitor cells can be isolated and expanded from orbital adipose tissues. Further characterization of their mesodermal or neuroectodermal origin might enhance clinical outcome when used as a source of autologous stem cells for ocular surface regeneration.
View details for DOI 10.1167/iovs.14-14441
View details for Web of Science ID 000343145500020
View details for PubMedID 24994870
View details for PubMedCentralID PMC4123896
Regulation of Intrinsic Axon Growth Ability at Retinal Ganglion Cell Growth Cones
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE
2014; 55 (7): 4369-4377
Mammalian central nervous system neurons fail to regenerate after injury or disease, in part due to a progressive loss in intrinsic axon growth ability after birth. Whether lost axon growth ability is due to limited growth resources or to changes in the axonal growth cone is unknown.Static and time-lapse images of purified retinal ganglion cells (RGCs) were analyzed for axon growth rate and growth cone morphology and dynamics without treatment and after manipulating Kruppel-like transcription factor (KLF) expression or applying mechanical tension.Retinal ganglion cells undergo a developmental switch in growth cone dynamics that mirrors the decline in postnatal axon growth rates, with increased filopodial adhesion and decreased lamellar protrusion area in postnatal axonal growth cones. Moreover, expressing growth-suppressive KLF4 or growth-enhancing KLF6 transcription factors elicits similar changes in postnatal growth cones that correlate with axon growth rates. Postnatal RGC axon growth rate is not limited by an inability to achieve axon growth rates similar to embryonic RGCs; indeed, postnatal axons support elongation rates up to 100-fold faster than postnatal axonal growth rates. Rather, the intrinsic capacity for rapid axon growth is due to both growth cone pausing and retraction, as well as to a slightly decreased ability to achieve rapid instantaneous rates of forward progression. Finally, we observed that RGC axon and dendrite growth are regulated independently in vitro.Together, these data support the hypothesis that intrinsic axon growth rate is regulated by an axon-specific growth program that differentially regulates growth cone motility.
View details for DOI 10.1167/iovs.14-13882
View details for Web of Science ID 000339487000045
View details for PubMedID 24906860
View details for PubMedCentralID PMC4102390
Femtosecond Laser-Assisted Astigmatic Keratotomy for Postoperative Trabeculectomy-Induced Corneal Astigmatism
JOURNAL OF REFRACTIVE SURGERY
2014; 30 (7): 502-504
To describe a case of postoperative trabeculectomy-induced corneal astigmatism treated with femtosecond laser-assisted astigmatic keratotomy.After trabeculectomy, the patient demonstrated change in manifest refraction from -0.5 diopters preoperatively to mixed astigmatism of -3.5 + 5.25@100 postoperatively and a decrease in uncorrected distance visual acuity from 20/60 preoperatively to 20/200 at 1 month postoperatively. Because the patient was intolerant to spectacle use, she underwent femtosecond laser-assisted astigmatic keratotomy.After astigmatic keratotomy there was improvement in corneal topographic astigmatism from 4.15 to 0.81 diopters with uncorrected distance visual acuity of 20/60(-2) and manifest refraction of -0.75 + 1.0@90 at 3 months postoperatively. There were no intraoperative or postoperative complications.Femtosecond laser-assisted astigmatic keratotomy may be considered in eyes with postoperative trabeculectomy-induced mixed astigmatism.
View details for DOI 10.3928/1081597X-20140527-01
View details for Web of Science ID 000338763000013
View details for PubMedID 24892377
Regulating Set-beta's Subcellular Localization Toggles Its Function between Inhibiting and Promoting Axon Growth and Regeneration
JOURNAL OF NEUROSCIENCE
2014; 34 (21): 7361-7374
The failure of the CNS neurons to regenerate axons after injury or stroke is a major clinical problem. Transcriptional regulators like Set-β are well positioned to regulate intrinsic axon regeneration capacity, which declines developmentally in maturing CNS neurons. Set-β also functions at cellular membranes and its subcellular localization is disrupted in Alzheimer's disease, but many of its biological mechanisms have not been explored in neurons. We found that Set-β was upregulated postnatally in CNS neurons, and was primarily localized to the nucleus but was also detected in the cytoplasm and adjacent to the plasma membrane. Remarkably, nuclear Set-β suppressed, whereas Set-β localized to cytoplasmic membranes promoted neurite growth in rodent retinal ganglion cells and hippocampal neurons. Mimicking serine 9 phosphorylation, as found in Alzheimer's disease brains, delayed nuclear import and furthermore blocked the ability of nuclear Set-β to suppress neurite growth. We also present data on gene regulation and protein binding partner recruitment by Set-β in primary neurons, raising the hypothesis that nuclear Set-β may preferentially regulate gene expression whereas Set-β at cytoplasmic membranes may regulate unique cofactors, including PP2A, which we show also regulates axon growth in vitro. Finally, increasing recruitment of Set-β to cellular membranes promoted adult rat optic nerve axon regeneration after injury in vivo. Thus, Set-β differentially regulates axon growth and regeneration depending on subcellular localization and phosphorylation.
View details for DOI 10.1523/JNEUROSCI.3658-13.2014
View details for Web of Science ID 000336895200030
View details for PubMedID 24849368
View details for PubMedCentralID PMC4028506
ACUTE RETINAL PIGMENT EPITHELIUM DETACHMENTS AFTER PHOTOCOAGULATION
RETINA-THE JOURNAL OF RETINAL AND VITREOUS DISEASES
2014; 34 (4): 749-760
To characterize the morphology of patterned scanning laser (PASCAL) panretinal photocoagulation.In this prospective cohort study, patients with proliferative diabetic retinopathy or severe nonproliferative diabetic retinopathy with high-risk characteristics, who were treated with PASCAL panretinal photocoagulation as part of their indicated clinical course, were serially imaged with spectral domain optical coherence tomography. Thirty eyes of 25 patients were studied from 1 hour to 21 weeks after laser treatment.Over a quarter (26.1%) of all treatment spots were imaged by spectral domain optical coherence tomography 1 hour after PASCAL panretinal photocoagulation. At 1 hour (±30 minutes) after PASCAL treatment, spectral domain optical coherence tomography demonstrated retinal pigment epithelium detachment in 23 of 27 eyes (85.2%) and in 36.1% of all imaged laser spots. Detachments occurred preferentially at the photocoagulation edges in 48.4% of pigment epithelium detachments (PEDs). Linear regression analysis revealed that average laser power (Pearson's r = 0.671, P < 0.001) and average laser energy (Pearson's r = 0.588, P = 0.001) were significantly associated with PEDs observed 1 hour after treatment. Pigment epithelium detachments occurred at a rate of 9.2% ± 4.9% at an average power of 0 mW to 400 mW, 37.8% ± 9.5% at 401 mW to 800 mW, 42.1% ± 5.6% at 801 mW to 1,200 mW, and 53.6% ± 5.7% at >1,200 mW. By a 1-week follow-up, no PEDs were observed, and the retinal pigment epithelium appeared morphologically similar to its preoperative structure by 3 weeks. Patient characteristics (study eye, sex, race, diagnosis, age, preoperative blood glucose, hemoglobin A1C, duration of diabetes, and body mass index) and other PASCAL parameters (number of laser applications, spot size, pulse duration, and average laser fluence) were not significantly associated with PEDs.Retinal pigment epithelium detachment occurs 1 hour after PASCAL treatment over a wide range of laser settings. Laser power and energy are positively correlated with the occurrence of PEDs, which are no longer observed by 1-week follow-up. Future studies might examine various acute posttreatment time points and directly compare the morphology of PASCAL burns with that of longer pulse-duration laser modalities.
View details for Web of Science ID 000336960500023
View details for PubMedID 24013258
Retinal repair with induced pluripotent stem cells
2014; 163 (4): 377-386
Retinal degeneration such as age-related macular degeneration and other inherited forms, such as Stargardt's disease and retinitis pigmentosa, and optic neuropathies including glaucoma and ischemic optic neuropathy are major causes of vision loss and blindness worldwide. Damage to retinal pigment epithelial cells and photoreceptors in the former, and to retinal ganglion cell axons in the optic nerve and their cell bodies in the retina in the latter diseases lead to the eventual death of these retinal cells, and in humans there is no endogenous replacement or repair. Cell replacement therapies provide 1 avenue to restore function in these diseases, particularly in the case of retinal repair, although there are considerable issues to overcome, including the differentiation and integration of the transplanted cells. What stem cell sources could be used for such therapies? One promising source is induced pluripotent stem cells (iPSCs), which could be drawn from an individual patient needing therapy, or generated and banked from select donors. We review developing research in the use of iPSCs for retinal cell replacement therapy.
View details for DOI 10.1016/j.trsl.2013.11.002
View details for Web of Science ID 000334648500011
View details for PubMedID 24291154
View details for PubMedCentralID PMC4073787
Survival and Integration of Developing and Progenitor-Derived Retinal Ganglion Cells Following Transplantation
2014; 23 (7): 855-872
There is considerable interest in transplanting stem cells or progenitors into the injured nervous system and enhancing their differentiation into mature, integrated, functional neurons. Little is known, however, about what intrinsic or extrinsic signals control the integration of differentiated neurons, either during development or in the adult. Here we ask whether purified, postmitotic, differentiated retinal ganglion cells (RGCs) directly isolated from rat retina or derived from in vitro-differentiated retinal progenitor cells can survive, migrate, extend neurites, and form morphologic synapses in a host retina, in vivo and ex vivo. We found that acutely purified primary and in vitro-differentiated RGCs survive transplantation and migrate into deeper retinal layers, including into their normal environment, the ganglion cell layer (GCL). Transplanted RGCs from a wide range of developmental ages, but not from adults, were capable of extending lengthy neurites in the normal and injured adult rat retina ex vivo and to a lesser degree after transplantation in vivo. We have also demonstrated that RGCs may be differentiated and purified from retinal precursor cultures and that they share many of the same cell biological properties as primary RGCs. We have established that progenitor-derived RGCs have similar capacity for integration as developing primary RGCs but appear to form a lower number of presynaptic punctae. This work provides insight for further understanding of the integration of developing RGCs into their normal environment and following injury.
View details for DOI 10.3727/096368913X667024
View details for Web of Science ID 000337989700006
View details for PubMedID 23636049
Fuchs endothelial corneal dystrophy: clinical characteristics of surgical and nonsurgical patients.
Clinical ophthalmology (Auckland, N.Z.)
2014; 8: 1761-1766
To review the patient and clinical characteristics of patients with Fuchs endothelial corneal dystrophy (FECD).Review of records for every patient who presented to the Bascom Palmer Eye Institute between 2003 and 2009 whose visit was coded for endothelial corneal dystrophy (International Classification of Diseases, Ninth Revision [ICD9] 371.57), bullous keratopathy (ICD9 371.23), or who underwent a corneal surgery with or without cataract extraction. Demographic, clinical, and ancillary testing data were collected from the time of presentation, diagnosis, and follow-up, and the use, timing, and type of surgical interventions was documented, with 6-month and final visual acuities recorded.A total of 2,370 charts were included in this study, of which 966 patients had a diagnosis of FECD. Of these, 197 patients (21%) received a corneal transplantation procedure. The surgery most often performed was penetrating keratoplasty with or without cataract extraction (66%), followed by endothelial keratoplasty with or without cataract extraction (34%). The risk factors for surgery include worse visual acuity at presentation (20/60 Snellen visual acuity in surgical patients versus 20/40 Snellen visual acuity in nonsurgical patients, P<0.001), greater average central corneal thickness (635 μm versus 592 μm, P<0.001), loss of visual acuity over time (two lines lost versus zero lines lost, P<0.001), increasing age (P<0.001), and male sex (P=0.008). Over half of patients (52%) did not receive surgery despite poor vision.During this time period, FECD did not have a consistent pattern for management or treatment, and despite advances in surgical techniques, most patients were still managed without surgery.
View details for DOI 10.2147/OPTH.S68217
View details for PubMedID 25228793
View details for PubMedCentralID PMC4164288
- How to measure vision in glaucoma. JAMA ophthalmology 2013; 131 (12): 1563-4
Amacrine Cell Subtypes Differ in Their Intrinsic Neurite Growth Capacity
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE
2013; 54 (12): 7603-7613
Amacrine cell neurite patterning has been extensively studied in vivo, and more than 30 subpopulations with varied morphologies have been identified in the mammalian retina. It is not known, however, whether the complex amacrine cell morphology is determined intrinsically, is signaled by extrinsic cues, or both.Here we purified rat amacrine cell subpopulations away from their retinal neighbors and glial-derived factors to ask questions about their intrinsic neurite growth ability. In defined medium strongly trophic for amacrine cells in vitro, we characterized survival and neurite growth of amacrine cell subpopulations defined by expression of specific markers.We found that a series of amacrine cell subtype markers are developmentally regulated, turning on through early postnatal development. Subtype marker expression was observed in similar fractions of cultured amacrine cells as was observed in vivo, and was maintained with time in culture. Overall, amacrine cell neurite growth followed principles very similar to those in postnatal retinal ganglion cells, but embryonic retinal ganglion cells demonstrated different features, relating to their rapid axon growth. Surprisingly, the three subpopulations of amacrine cells studied in vitro recapitulated quantitatively and qualitatively the varied morphologies they have in vivo.Our data suggest that cultured amacrine cells maintain intrinsic fidelity to their identified in vivo subtypes, and furthermore, that cell-autonomous, intrinsic factors contribute to the regulation of neurite patterning.
View details for DOI 10.1167/iovs.13-12691
View details for Web of Science ID 000327949700053
View details for PubMedID 24130183
View details for PubMedCentralID PMC3832218
A tunable synthetic hydrogel system for culture of retinal ganglion cells and amacrine cells
2013; 9 (8): 7622-7629
The central nervous system consists of complex groups of individual cells that receive electrical, chemical and physical signals from their local environment. Standard in vitro cell culture methods rely on two-dimensional (2-D) substrates that poorly simulate in vivo neural architecture. Neural cells grown in three-dimensional (3-D) culture systems may provide an opportunity to study more accurate representations of the in vivo environment than 2-D cultures. Furthermore, each specific type of neuron depends on discrete compositions and physical properties of their local environment. Previously, we developed a library of hydrogels composed of poly(ethylene glycol) and poly(l-lysine) which exhibit a wide range of mechanical properties. Here, we identified specific scaffolds from this library that readily support the survival, migration and neurite outgrowth of purified retinal ganglion cells and amacrine cells. These data address important biological questions about the interaction of neurons with the physical and chemical properties of their local environment and provide further insight for engineering neural tissue for cell-replacement therapies following injury.
View details for DOI 10.1016/j.actbio.2013.04.048
View details for Web of Science ID 000322207700002
View details for PubMedID 23648573
View details for PubMedCentralID PMC3722500
Tissue engineering the retinal ganglion cell nerve fiber layer
2013; 34 (17): 4242-4250
Retinal degenerative diseases, such as glaucoma and macular degeneration, affect millions of people worldwide and ultimately lead to retinal cell death and blindness. Cell transplantation therapies for photoreceptors demonstrate integration and restoration of function, but transplantation into the ganglion cell layer is more complex, requiring guidance of axons from transplanted cells to the optic nerve head in order to reach targets in the brain. Here we create a biodegradable electrospun (ES) scaffold designed to direct the growth of retinal ganglion cell (RGC) axons radially, mimicking axon orientation in the retina. Using this scaffold we observed an increase in RGC survival and no significant change in their electrophysiological properties. When analyzed for alignment, 81% of RGCs were observed to project axons radially along the scaffold fibers, with no difference in alignment compared to the nerve fiber layer of retinal explants. When transplanted onto retinal explants, RGCs on ES scaffolds followed the radial pattern of the host retinal nerve fibers, whereas RGCs transplanted directly grew axons in a random pattern. Thus, the use of this scaffold as a cell delivery device represents a significant step towards the use of cell transplant therapies for the treatment of glaucoma and other retinal degenerative diseases.
View details for DOI 10.1016/j.biomaterials.2013.02.027
View details for Web of Science ID 000317700400005
View details for PubMedID 23489919
View details for PubMedCentralID PMC3608715
Nanotechnology and glaucoma: little particles for a big disease
CURRENT OPINION IN OPHTHALMOLOGY
2013; 24 (2): 130-135
Current medical treatments designed to halt the progressive loss of retinal ganglion cells (RGCs) in glaucoma are limited by low bioavailability to target tissues and lack of patient adherence to frequent dosing regimens. For a certain percentage of patients with glaucoma, reducing intraocular pressure (IOP) does not stop disease progression, motivating the search for new therapeutic targets and delivery systems.The emerging science of nanoparticles has the potential to address the current limitations of glaucoma therapy by improving drug bioavailability, exploiting IOP-independent targets such as RGC neuroprotection, and optimizing gene therapy as a more permanent treatment for glaucoma.We review the recent advances in nanoparticle-based glaucoma therapy with a focus on drug delivery to the eye, as well as novel applications including gene therapy.
View details for DOI 10.1097/ICU.0b013e32835cfe92
View details for Web of Science ID 000317039900007
View details for PubMedID 23287105
A Novel Rodent Model of Posterior Ischemic Optic Neuropathy
2013; 131 (2): 194-204
To develop a reliable, reproducible rat model of posterior ischemic optic neuropathy (PION) and study the cellular responses in the optic nerve and retina.Posterior ischemic optic neuropathy was induced in adult rats by photochemically induced ischemia. Retinal and optic nerve vasculature was examined by fluorescein isothiocyanate–dextran extravasation. Tissue sectioning and immunohistochemistry were used to investigate the pathologic changes. Retinal ganglion cell survival at different times after PION induction, with or without neurotrophic application, was quantified by fluorogold retrograde labeling.Optic nerve injury was confirmed after PION induction, including local vascular leakage, optic nerve edema, and cavernous degeneration. Immunostaining data revealed microglial activation and focal loss of astrocytes, with adjacent astrocytic hypertrophy. Up to 23%, 50%, and 70% retinal ganglion cell loss was observed at 1 week, 2 weeks, and 3 weeks, respectively, after injury compared with a sham control group. Experimental treatment by brain-derived neurotrophic factor and ciliary neurotrophic factor remarkably prevented retinal ganglion cell loss in PION rats. At 3 weeks after injury, more than 40% of retinal ganglion cells were saved by the application of neurotrophic factors.Rat PION created by photochemically induced ischemia is a reproducible and reliable animal model for mimicking the key features of human PION.The correspondence between the features of this rat PION model to those of human PION makes it an ideal model to study the pathophysiologic course of the disease, most of which remains to be elucidated. Furthermore, it provides an optimal model for testing therapeutic approaches for optic neuropathies.
View details for DOI 10.1001/2013.jamaophthalmol.271
View details for Web of Science ID 000316684400010
View details for PubMedID 23544206
View details for PubMedCentralID PMC4885914
beta 1 Integrin-Focal Adhesion Kinase (FAK) Signaling Modulates Retinal Ganglion Cell (RGC) Survival
2012; 7 (10)
Extracellular matrix (ECM) integrity in the central nervous system (CNS) is essential for neuronal homeostasis. Signals from the ECM are transmitted to neurons through integrins, a family of cell surface receptors that mediate cell attachment to ECM. We have previously established a causal link between the activation of the matrix metalloproteinase-9 (MMP-9), degradation of laminin in the ECM of retinal ganglion cells (RGCs), and RGC death in a mouse model of retinal ischemia-reperfusion injury (RIRI). Here we investigated the role of laminin-integrin signaling in RGC survival in vitro, and after ischemia in vivo. In purified primary rat RGCs, stimulation of the β1 integrin receptor with laminin, or agonist antibodies enhanced RGC survival in correlation with activation of β1 integrin's major downstream regulator, focal adhesion kinase (FAK). Furthermore, β1 integrin binding and FAK activation were required for RGCs' survival response to laminin. Finally, in vivo after RIRI, we observed an up-regulation of MMP-9, proteolytic degradation of laminin, decreased RGC expression of β1 integrin, FAK and Akt dephosphorylation, and reduced expression of the pro-survival molecule bcl-xL in the period preceding RGC apoptosis. RGC death was prevented, in the context of laminin degradation, by maintaining β1 integrin activation with agonist antibodies. Thus, disruption of homeostatic RGC-laminin interaction and signaling leads to cell death after retinal ischemia, and maintaining integrin activation may be a therapeutic approach to neuroprotection.
View details for DOI 10.1371/journal.pone.0048332
View details for Web of Science ID 000310600500108
View details for PubMedID 23118988
View details for PubMedCentralID PMC3485184
Mitochondrial Dynamics Regulate Growth Cone Motility, Guidance, and Neurite Growth Rate in Perinatal Retinal Ganglion Cells In Vitro
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE
2012; 53 (11): 7402-7411
Retinal ganglion cell (RGC) death and failed axonal regeneration after trauma or disease, including glaucomatous and mitochondrial optic neuropathies, are linked increasingly to dysfunctional mitochondrial dynamics. However, how mitochondrial dynamics influence axon growth largely is unstudied. We examined intrinsic mitochondrial organization in embryonic and postnatal RGCs and the roles that mitochondrial dynamics have in regulating neurite growth and guidance.RGCs were isolated from embryonic day 20 (E20) or postnatal days 5 to 7 (P5-7) Sprague-Dawley rats by anti-Thy1 immunopanning. After JC-1 loading, mitochondria were analyzed in acutely purified RGCs by flow cytometry and in RGC neurites by fluorescence microscopy. Intrinsic axon growth was modulated by overexpressing Krüppel-like family (KLF) transcription factors, or mitochondrial dynamics were altered by inhibiting dynamin related protein-1 (DRP-1) pharmacologically or by overexpressing mitofusin-2 (Mfn-2). Mitochondrial organization, neurite growth, and growth cone motility and guidance were analyzed.Mitochondrial dynamics and function are regulated developmentally in acutely purified RGCs and in nascent RGC neurites. Mitochondrial dynamics are modulated differentially by KLFs that promote or suppress growth. Acutely inhibiting mitochondrial fission reversibly suppressed axon growth and lamellar extension. Inhibiting DRP-1 or overexpressing Mfn-2 altered growth cone responses to chondroitin sulfate proteoglycan, netrin-1, and fibronectin.These results support the hypothesis that mitochondria locally modulate signaling in the distal neurite and growth cone to affect the direction and the rate of neurite growth.
View details for DOI 10.1167/iovs.12-10298
View details for Web of Science ID 000310589900094
View details for PubMedID 23049086
View details for PubMedCentralID PMC3484733
Scaffolds and stem cells: delivery of cell transplants for retinal degenerations.
Expert review of ophthalmology
2012; 7 (5): 459-470
Retinal degenerations and optic neuropathies often lead to death of photoreceptors or retinal ganglion cells, respectively. Stem cell therapies are showing promise for these diseases in preclinical models and are beginning to transition into human trials, but cell delivery and integration remain major challenges. Focusing on photoreceptor- and progenitor-directed approaches, in this article, the authors review how advances in tissue engineering and cell scaffold design are enhancing cell therapies for retinal degeneration.
View details for PubMedID 23585772
Role of electrical activity in promoting neural repair
2012; 519 (2): 134-137
The nervous system communicates in a language of electrical activities. The motivation to replace function lost through injury or disease through electrical prostheses has gained traction through steady advances in basic and translational science addressing the interface between electrical prostheses and the nervous system. Recent experiments suggest that electrical activity, signaling through specific molecular pathways, promotes neuronal survival and regeneration. Such data suggests that electrical prostheses, in addition to replacing lost function, may slow underlying degenerative disease or induce regenerative response. Here we review these data with a focus on retinal neurons, and discuss current efforts to translate this effect of electrical activity into clinically applicable treatments.
View details for DOI 10.1016/j.neulet.2012.02.003
View details for Web of Science ID 000306146800008
View details for PubMedID 22342908
View details for PubMedCentralID PMC3360133
A chemical genetic approach identifies piperazine antipsychotics as promoters of CNS neurite growth on inhibitory substrates
MOLECULAR AND CELLULAR NEUROSCIENCE
2012; 50 (2): 125-135
Injury to the central nervous system (CNS) can result in lifelong loss of function due in part to the regenerative failure of CNS neurons. Inhibitory proteins derived from myelin and the astroglial scar are major barriers for the successful regeneration of injured CNS neurons. Previously, we described the identification of a novel compound, F05, which promotes neurite growth from neurons challenged with inhibitory substrates in vitro, and promotes axonal regeneration in vivo (Usher et al., 2010). To identify additional regeneration-promoting compounds, we used F05-induced gene expression profiles to query the Broad Institute Connectivity Map, a gene expression database of cells treated with >1300 compounds. Despite no shared chemical similarity, F05-induced changes in gene expression were remarkably similar to those seen with a group of piperazine phenothiazine antipsychotics (PhAPs). In contrast to antipsychotics of other structural classes, PhAPs promoted neurite growth of CNS neurons challenged with two different glial derived inhibitory substrates. Our pharmacological studies suggest a mechanism whereby PhAPs promote growth through antagonism of calmodulin signaling, independent of dopamine receptor antagonism. These findings shed light on mechanisms underlying neurite-inhibitory signaling, and suggest that clinically approved antipsychotic compounds may be repurposed for use in CNS injured patients.
View details for DOI 10.1016/j.mcn.2012.04.008
View details for Web of Science ID 000306304300001
View details for PubMedID 22561309
View details for PubMedCentralID PMC3383383
Soluble Adenylyl Cyclase Activity Is Necessary for Retinal Ganglion Cell Survival and Axon Growth
JOURNAL OF NEUROSCIENCE
2012; 32 (22): 7734-7744
cAMP is a critical second messenger mediating activity-dependent neuronal survival and neurite growth. We investigated the expression and function of the soluble adenylyl cyclase (sAC, ADCY10) in CNS retinal ganglion cells (RGCs). We found sAC protein expressed in multiple RGC compartments including the nucleus, cytoplasm and axons. sAC activation increased cAMP above the level seen with transmembrane adenylate cyclase (tmAC) activation. Electrical activity and bicarbonate, both physiologic sAC activators, significantly increased survival and axon growth, whereas pharmacologic or siRNA-mediated sAC inhibition dramatically decreased RGC survival and axon growth in vitro, and survival in vivo. Conversely, RGC survival and axon growth were unaltered in RGCs from AC1/AC8 double knock-out mice or after specifically inhibiting tmACs. These data identify a novel sAC-mediated cAMP signaling pathway regulating RGC survival and axon growth, and suggest new neuroprotective or regenerative strategies based on sAC modulation.
View details for DOI 10.1523/JNEUROSCI.5288-11.2012
View details for Web of Science ID 000304627100031
View details for PubMedID 22649251
View details for PubMedCentralID PMC3372574
Kruppel-like Factor 7 engineered for transcriptional activation promotes axon regeneration in the adult corticospinal tract
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2012; 109 (19): 7517-7522
Axon regeneration in the central nervous system normally fails, in part because of a developmental decline in the intrinsic ability of CNS projection neurons to extend axons. Members of the KLF family of transcription factors regulate regenerative potential in developing CNS neurons. Expression of one family member, KLF7, is down-regulated developmentally, and overexpression of KLF7 in cortical neurons in vitro promotes axonal growth. To circumvent difficulties in achieving high neuronal expression of exogenous KLF7, we created a chimera with the VP16 transactivation domain, which displayed enhanced neuronal expression compared with the native protein while maintaining transcriptional activation and growth promotion in vitro. Overexpression of VP16-KLF7 overcame the developmental loss of regenerative ability in cortical slice cultures. Adult corticospinal tract (CST) neurons failed to up-regulate KLF7 in response to axon injury, and overexpression of VP16-KLF7 in vivo promoted both sprouting and regenerative axon growth in the CST of adult mice. These findings identify a unique means of promoting CST axon regeneration in vivo by reengineering a developmentally down-regulated, growth-promoting transcription factor.
View details for DOI 10.1073/pnas.1120684109
View details for Web of Science ID 000304090600078
View details for PubMedID 22529377
View details for PubMedCentralID PMC3358880
Glaucoma 2.0: Neuroprotection, Neuroregeneration, Neuroenhancement
2012; 119 (5): 979-986
Glaucoma is a progressive neurodegenerative disease of retinal ganglion cells (RGCs) associated with characteristic axon degeneration in the optic nerve. Clinically, our only method of slowing glaucomatous loss of vision is to reduce intraocular pressure (IOP), but lowering IOP is only partially effective and does not address the underlying susceptibility of RGCs to degeneration. We review the recent steps forward in our understanding of the pathophysiology of glaucoma and discuss how this understanding has given us a next generation of therapeutic targets by which to maintain RGC survival, protect or rebuild RGC connections in the retina and brain, and enhance RGC function.
View details for DOI 10.1016/j.ophtha.2011.11.003
View details for Web of Science ID 000303399800014
View details for PubMedID 22349567
View details for PubMedCentralID PMC3343191
Investigation of nanoparticles using magnetic resonance imaging after intravitreal injection
CLINICAL AND EXPERIMENTAL OPHTHALMOLOGY
2012; 40 (1): 100-107
Magnetic nanoparticles may be used for focal delivery for cells, plasmids or drugs, and other applications. Here we asked whether magnetic nanoparticles could be detected in vivo at different time points after intravitreal injection by magnetic resonance imaging.Adult Sprague-Dawley rats received intravitreal injections of 50-nm or 4-µm magnetic particles into the left eye, with an equal volume of phosphate-buffered saline into the right eye (as controls). Animals were examined by magnetic resonance imaging at 1 h, 1 day and 5 weeks after injection. Eyes, brain, liver, spleen and kidney were also imaged with high-resolution ex vivo magnetic resonance imaging scanning.In vivo magnetic resonance imaging at the 1 h and 1 day time points more clearly detected magnetic particles in the 4 µm group compared with the 50-nm group, although 50-nm magnetic nanoparticles were easily visualized with high-resolution magnetic resonance imaging ex vivo. Five weeks after intravitreal injection magnetic resonance imaging clearly detected 4-µm particles inside the eye, but by this time point the 50-nm magnetic nanoparticles could not be detected by either in vivo or ex vivo high-resolution magnetic resonance imaging. No magnetic particles were detected in any other organ.Magnetic resonance imaging could be used to track magnetic nanoparticles in the eye with the dosing selected for this study. Clearance varies by size, with 50-nm magnetic nanoparticles cleared more quickly than 4-µm particles. Thus, nanoparticles may provide advantages over micron-scale particles when considering risks associated with long-term persistence.
View details for DOI 10.1111/j.1442-9071.2011.02651.x
View details for Web of Science ID 000300000800035
View details for PubMedID 21745263
Epigenetic regulation of axon and dendrite growth.
Frontiers in molecular neuroscience
2012; 5: 24-?
Neuroregenerative therapies for central nervous system (CNS) injury, neurodegenerative disease, or stroke require axons of damaged neurons to grow and re-innervate their targets. However, mature mammalian CNS neurons do not regenerate their axons, limiting recovery in these diseases. Although neurons' intrinsic capacity for axon growth may depend in part on the panoply of expressed transcription factors, epigenetic factors such as the accessibility of DNA and organization of chromatin are required for downstream genes to be transcribed. Thus, a potential approach to overcoming regenerative failure focuses on the epigenetic mechanisms regulating regenerative gene expression in the CNS. Here we review molecular mechanisms regulating the epigenetic state of DNA through chromatin modifications, their implications for regulating axon and dendrite growth, and important new directions for this field of study.
View details for DOI 10.3389/fnmol.2012.00024
View details for PubMedID 22403528
View details for PubMedCentralID PMC3290832
- Epigenetic regulation of axon and dendrite growth FRONTIERS IN MOLECULAR NEUROSCIENCE 2012; 5
The Role of Serotonin in Axon and Dendrite Growth
AXON GROWTH AND REGENERATION, PT 2
2012; 106: 105-126
The neurotransmitter serotonin (5-hydroxytryptamine, 5-HT) plays multiple roles in the enteric, peripheral, and central nervous systems (CNS). Although its most prominent biological function is as a signal transmission messenger from pre- to postsynaptic neurons, other roles such as shaping brain development and regulating neurite growth have also been described. Here, we review the less well-studied role of 5-HT as a modulator of neurite growth. 5-HT has been shown to regulate neurite growth in multiple systems and species, including in the mammalian CNS. 5-HT predominantly appears to suppress neurite growth, but depending on the model system and 5-HT receptor subtype, in rare cases, it may promote neurite outgrowth and elongation. Failure of axon regeneration in the adult mammalian CNS is a major problem in multiple diseases, and understanding how 5-HT receptors signal opposing effects on neurite growth may lead to novel neuroregenerative therapies, by targeting either 5-HT receptors or their downstream signaling pathways.
View details for DOI 10.1016/B978-0-12-407178-0.00005-3
View details for Web of Science ID 000314132900004
View details for PubMedID 23211461
Signaling Endosomes and Growth Cone Motility in Axon Regeneration
AXON GROWTH AND REGENERATION, PT 2
2012; 106: 35-73
During development and regeneration, growth cones guide neurites to their targets by altering their motility in response to extracellular guidance cues. One class of cues critical to nervous system development is the neurotrophins. Neurotrophin binding to their cognate receptors stimulates their endocytosis into signaling endosomes. Current data indicate that the spatiotemporal localization of signaling endosomes can direct diverse processes regulating cell motility, including membrane trafficking, cytoskeletal remodeling, adhesion dynamics, and local translation. Recent experiments manipulating signaling endosome localization in neuronal growth cones support these views and place the neurotrophin signaling endosome in a central role regulating growth cone motility during axon growth and regeneration.
View details for DOI 10.1016/B978-0-12-407178-0.00003-X
View details for Web of Science ID 000314132900002
View details for PubMedID 23211459
Multiple Transcription Factor Families Regulate Axon Growth and Regeneration
2011; 71 (12): 1186-1211
Understanding axon regenerative failure remains a major goal in neuroscience, and reversing this failure remains a major goal for clinical neurology. Although an inhibitory central nervous system environment clearly plays a role, focus on molecular pathways within neurons has begun to yield fruitful insights. Initial steps forward investigated the receptors and signaling pathways immediately downstream of environmental cues, but recent work has also shed light on transcriptional control mechanisms that regulate intrinsic axon growth ability, presumably through whole cassettes of gene target regulation. Here we will discuss transcription factors that regulate neurite growth in vitro and in vivo, including p53, SnoN, E47, cAMP-responsive element binding protein (CREB), signal transducer and activator of transcription 3 (STAT3), nuclear factor of activated T cell (NFAT), c-Jun activating transcription factor 3 (ATF3), sex determining region Ybox containing gene 11 (Sox11), nuclear factor κ-light chain enhancer of activated B cells (NFκB), and Krüppel-like factors (KLFs). Revealing the similarities and differences among the functions of these transcription factors may further our understanding of the mechanisms of transcriptional regulation in axon growth and regeneration.
View details for DOI 10.1002/dneu.20934
View details for Web of Science ID 000298234400005
View details for PubMedID 21674813
View details for PubMedCentralID PMC3212623
Nanoparticle-mediated signaling endosome localization regulates growth cone motility and neurite growth
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2011; 108 (47): 19042-19047
Understanding neurite growth regulation remains a seminal problem in neurobiology. During development and regeneration, neurite growth is modulated by neurotrophin-activated signaling endosomes that transmit regulatory signals between soma and growth cones. After injury, delivering neurotrophic therapeutics to injured neurons is limited by our understanding of how signaling endosome localization in the growth cone affects neurite growth. Nanobiotechnology is providing new tools to answer previously inaccessible questions. Here, we show superparamagnetic nanoparticles (MNPs) functionalized with TrkB agonist antibodies are endocytosed into signaling endosomes by primary neurons that activate TrkB-dependent signaling, gene expression and promote neurite growth. These MNP signaling endosomes are trafficked into nascent and existing neurites and transported between somas and growth cones in vitro and in vivo. Manipulating MNP-signaling endosomes by a focal magnetic field alters growth cone motility and halts neurite growth in both peripheral and central nervous system neurons, demonstrating signaling endosome localization in the growth cone regulates motility and neurite growth. These data suggest functionalized MNPs may be used as a platform to study subcellular organelle localization and to deliver nanotherapeutics to treat injury or disease in the central nervous system.
View details for DOI 10.1073/pnas.1019624108
View details for Web of Science ID 000297249800041
View details for PubMedID 22065745
View details for PubMedCentralID PMC3223462
- Immunology. Neuroimmune communication. Science (New York, N.Y.) 2011; 334 (6052): 47-8
Kruppel-like transcription factors in the nervous system: Novel players in neurite outgrowth and axon regeneration
MOLECULAR AND CELLULAR NEUROSCIENCE
2011; 47 (4): 233-243
The Krüppel-like family of transcription factors (KLFs) have been widely studied in proliferating cells, though very little is known about their role in post-mitotic cells, such as neurons. We have recently found that the KLFs play a role in regulating intrinsic axon growth ability in retinal ganglion cells (RGCs), a type of central nervous system (CNS) neuron. Previous KLF studies in other cell types suggest that there may be cell-type specific KLF expression patterns, and that their relative expression allows them to compete for binding sites, or to act redundantly to compensate for another's function. With at least 15 of 17 KLF family members expressed in neurons, it will be important for us to determine how this complex family functions to regulate the intricate gene programs of axon growth and regeneration. By further characterizing the mechanisms of the KLF family in the nervous system, we may better understand how they regulate neurite growth and axon regeneration.
View details for DOI 10.1016/j.mcn.2011.05.005
View details for Web of Science ID 000293309500001
View details for PubMedID 21635952
View details for PubMedCentralID PMC3143062
Evaluation of Magnetic Micro- and Nanoparticle Toxicity to Ocular Tissues
2011; 6 (5)
Magnetic nanoparticles (MNPs) may be used for focal delivery of plasmids, drugs, cells, and other applications. Here we ask whether such particles are toxic to ocular structures.To evaluate the ocular toxicity of MNPs, we asked if either 50 nm or 4 µm magnetic particles affect intraocular pressure, corneal endothelial cell count, retinal morphology including both cell counts and glial activation, or photoreceptor function at different time points after injection. Sprague-Dawley rats (n = 44) were injected in the left eye with either 50 nm (3 µl, 1.65 mg) or 4 µm (3 µl, 1.69 mg) magnetic particles, and an equal volume of PBS into the right eye. Electroretinograms (ERG) were used to determine if MNPs induce functional changes to the photoreceptor layers. Enucleated eyes were sectioned for histology and immunofluorescence.Compared to control-injected eyes, MNPs did not alter IOP measurements. ERG amplitudes for a-waves were in the 100-250 µV range and b-waves were in the 500-600 µV range, with no significant differences between injected and non-injected eyes. Histological sectioning and immunofluorescence staining showed little difference in MNP-injected animals compared to control eyes. In contrast, at 1 week, corneal endothelial cell numbers were significantly lower in the 4 µm magnetic particle-injected eyes compared to either 50 nm MNP- or PBS-injected eyes. Furthermore, iron deposition was detected after 4 µm magnetic particle but not 50 nm MNP injection.Intravitreal or anterior chamber injections of MNPs showed little to no signs of toxicity on retinal structure, photoreceptor function or aqueous drainage in the eye. Our results suggest that MNPs are safe for intraocular use.
View details for DOI 10.1371/journal.pone.0017452
View details for Web of Science ID 000291052200003
View details for PubMedID 21637340
View details for PubMedCentralID PMC3102660
Foxn4 is required for retinal ganglion cell distal axon patterning
MOLECULAR AND CELLULAR NEUROSCIENCE
2011; 46 (4): 731-741
The regulation of retinal ganglion cell (RGC) axon growth and patterning in vivo is thought to be largely dependent on interactions with visual pathway and target cells. Here we address the hypothesis that amacrine cells, RGCs' presynaptic partners, regulate RGC axon growth or targeting. We asked whether amacrine cells play a role in RGC axon growth in vivo using Foxn4(-/-) mice, which have fewer amacrine cells, but a normal complement of RGCs. We found that Foxn4(-/-) mice have a similar reduction in most subtypes of amacrine cells examined. Remarkably, spontaneous retinal waves were not affected by the reduction of amacrine cells in the Foxn4(-/-) mice. There was, however, a developmental delay in the distribution of RGC projections to the superior colliculus. Furthermore, RGC axons failed to penetrate into the retinorecipient layers in the Foxn4(-/-) mice. Foxn4 is not expressed by RGCs and was not detectable in the superior colliculus itself. These findings suggest that amacrine cells are critical for proper RGC axon growth in vivo, and support the hypothesis that the amacrine cell-RGC interaction may contribute to the regulation of distal projections and axon patterning.
View details for DOI 10.1016/j.mcn.2011.02.004
View details for Web of Science ID 000289389100005
View details for PubMedID 21334440
View details for PubMedCentralID PMC3081519
Four Steps to Optic Nerve Regeneration
JOURNAL OF NEURO-OPHTHALMOLOGY
2010; 30 (4): 347-360
The failure of the optic nerve to regenerate after injury or in neurodegenerative disease remains a major clinical and scientific problem. Retinal ganglion cell (RGC) axons course through the optic nerve and carry all the visual information to the brain, but after injury, they fail to regrow through the optic nerve and RGC cell bodies typically die, leading to permanent loss of vision. There are at least 4 hurdles to overcome in preserving RGCs and regenerating their axons: 1) increase RGC survival, 2) overcome the inhibitory environment of the optic nerve, 3) enhance RGC intrinsic axon growth potential, and 4) optimize the mapping of RGC connections back into their targets in the brain.
View details for DOI 10.1097/WNO.0b013e3181e755af
View details for Web of Science ID 000284603900011
View details for PubMedID 21107123
Amacrine Cell Gene Expression and Survival Signaling: Differences from Neighboring Retinal Ganglion Cells
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE
2010; 51 (7): 3800-3812
PURPOSE. To describe how developing amacrine cells and retinal ganglion cells (RGCs) differ in survival signaling and global gene expression. METHODS. Amacrine cells were immunopurified and processed for gene microarray analysis. For survival studies, purified amacrine cells were cultured at low density in serum-free medium, with and without peptide trophic factors and survival pathway inhibitors. The differences in gene expression between amacrine cells and RGCs were analyzed by comparing the transcriptomes of these two cell types at the same developmental ages. RESULTS. The amacrine cell transcriptome was very dynamic during development. Amacrine cell gene expression was remarkably similar to that of RGCs, but differed in several gene ontologies, including polarity- and neurotransmission-associated genes. Unlike RGCs, amacrine cell survival in vitro was independent of cell density and the presence of exogenous trophic factors, but necessitated Erk activation via MEK1/2 and AKT signaling. Finally, comparison of the gene expression profile of amacrine cells and RGCs provided a list of polarity-associated candidate genes that may explain the inability of amacrine cells to differentiate axons and dendrites as RGCs do. CONCLUSIONS. Comparison of the gene expression profile between amacrine cells and RGCs may improve our understanding of why amacrine cells fail to differentiate axons and dendrites during retinal development and of what makes amacrine cells differ in their resistance to neurodegeneration. Switching RGCs to an amacrine cell-like state could help preserve their survival in neurodegenerative diseases like glaucoma, and amacrine cells could provide a ready source of replacement RGCs in such optic neuropathies.
View details for DOI 10.1167/iovs.09-4540
View details for Web of Science ID 000279047500063
View details for PubMedID 20445109
View details for PubMedCentralID PMC2904021
High content screening of cortical neurons identifies novel regulators of axon growth
MOLECULAR AND CELLULAR NEUROSCIENCE
2010; 44 (1): 43-54
Neurons in the central nervous system lose their intrinsic capacity for axon regeneration as they mature, and it is widely hypothesized that changes in gene expression are responsible. Testing this hypothesis and identifying the relevant genes has been challenging because hundreds to thousands of genes are developmentally regulated in CNS neurons, but only a small subset are likely relevant to axon growth. Here we used automated high content analysis (HCA) methods to functionally test 743 plasmids encoding developmentally regulated genes in neurite outgrowth assays using postnatal cortical neurons. We identified both growth inhibitors (Ephexin, Aldolase A, Solute Carrier 2A3, and Chimerin), and growth enhancers (Doublecortin, Doublecortin-like, Kruppel-like Factor 6, and CaM-Kinase II gamma), some of which regulate established growth mechanisms like microtubule dynamics and small GTPase signaling. Interestingly, with only one exception the growth-suppressing genes were developmentally upregulated, and the growth-enhancing genes downregulated. These data provide important support for the hypothesis that developmental changes in gene expression control neurite outgrowth, and identify potential new gene targets to promote neurite outgrowth.
View details for DOI 10.1016/j.mcn.2010.02.002
View details for Web of Science ID 000276588800004
View details for PubMedID 20159039
View details for PubMedCentralID PMC2890283
A Chemical Screen Identifies Novel Compounds That Overcome Glial-Mediated Inhibition of Neuronal Regeneration
JOURNAL OF NEUROSCIENCE
2010; 30 (13): 4693-4706
A major barrier to regeneration of CNS axons is the presence of growth-inhibitory proteins associated with myelin and the glial scar. To identify chemical compounds with the ability to overcome the inhibition of regeneration, we screened a novel triazine library, based on the ability of compounds to increase neurite outgrowth from cerebellar neurons on inhibitory myelin substrates. The screen produced four "hit compounds," which act with nanomolar potency on several different neuronal types and on several distinct substrates relevant to glial inhibition. Moreover, the compounds selectively overcome inhibition rather than promote growth in general. The compounds do not affect neuronal cAMP levels, PKC activity, or EGFR (epidermal growth factor receptor) activation. Interestingly, one of the compounds alters microtubule dynamics and increases microtubule density in both fibroblasts and neurons. This same compound promotes regeneration of dorsal column axons after acute lesions and potentiates regeneration of optic nerve axons after nerve crush in vivo. These compounds should provide insight into the mechanisms through which glial-derived inhibitors of regeneration act, and could lead to the development of novel therapies for CNS injury.
View details for DOI 10.1523/JNEUROSCI.0302-10.2010
View details for Web of Science ID 000276178000020
View details for PubMedID 20357120
View details for PubMedCentralID PMC2855497
Neurotrophic Effect of a Novel TrkB Agonist on Retinal Ganglion Cells
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE
2010; 51 (3): 1747-1754
Retinal ganglion cells (RGCs) die in glaucoma and virtually all optic neuropathies. Recently, novel tropomyosin-related kinase B (TrkB) monoclonal antibodies have been shown to activate TrkB receptors and exert neuroprotective and neurotrophic effects. In the present study, the authors examined the ability of one of them, 29D7, to elicit RGC survival and neurite growth both in culture and in vivo.RGCs from postnatal day (P)3 to P4 Sprague-Dawley rats were isolated by sequential immunopanning using a monoclonal antibody to Thy1. RGCs were cultured in serum-free defined medium in 96-well plates. RGC viability was assessed after 1 to 3 days by MTT assay. Activation of TrkB and downstream signaling molecules was confirmed by Western blot analysis. Intravitreal injections of 29D7 were performed after optic nerve axotomy, and subsequent RGC survival was quantified using beta-III tubulin immunostaining. Regeneration was assessed using retrograde fluorogold tracing in an optic nerve-peripheral nerve graft model.Similar to brain-derived neurotrophic factor (BDNF), the 29D7 antibody strongly promoted RGC survival and neurite growth in vitro compared with medium alone or control IgG. Forskolin, which weakly supported RGC survival on its own, potentiated the effect of 29D7. Intravitreal injection of 29D7 enhanced RGC survival but not regeneration in vivo 2 weeks after optic nerve injury.Together, these findings demonstrate the potential for antibody-mediated TrkB agonism as a potential therapeutic approach to enhance RGC survival after optic nerve injury. Further studies are needed to elucidate the mechanistic differences between this TrkB agonist and BDNF.
View details for DOI 10.1167/iovs.09-4450
View details for Web of Science ID 000275164300069
View details for PubMedID 19875669
View details for PubMedCentralID PMC2868417
KLF Family Members Regulate Intrinsic Axon Regeneration Ability
2009; 326 (5950): 298-301
Neurons in the central nervous system (CNS) lose their ability to regenerate early in development, but the underlying mechanisms are unknown. By screening genes developmentally regulated in retinal ganglion cells (RGCs), we identified Krüppel-like factor-4 (KLF4) as a transcriptional repressor of axon growth in RGCs and other CNS neurons. RGCs lacking KLF4 showed increased axon growth both in vitro and after optic nerve injury in vivo. Related KLF family members suppressed or enhanced axon growth to differing extents, and several growth-suppressive KLFs were up-regulated postnatally, whereas growth-enhancing KLFs were down-regulated. Thus, coordinated activities of different KLFs regulate the regenerative capacity of CNS neurons.
View details for DOI 10.1126/science.1175737
View details for Web of Science ID 000270599500045
View details for PubMedID 19815778
View details for PubMedCentralID PMC2882032
Electrical activity enhances neuronal survival and regeneration
JOURNAL OF NEURAL ENGINEERING
2009; 6 (5)
The failure of regeneration in the central nervous system (CNS) remains an enormous scientific and clinical challenge. After injury or in degenerative diseases, neurons in the adult mammalian CNS fail to regrow their axons and reconnect with their normal targets, and furthermore the neurons frequently die and are not normally replaced. While significant progress has been made in understanding the molecular basis for this lack of regenerative ability, a second approach has gained momentum: replacing lost neurons or lost connections with artificial electrical circuits that interface with the nervous system. In the visual system, gene therapy-based 'optogenetics' prostheses represent a competing technology. Now, the two approaches are converging, as recent data suggest that electrical activity itself, via the molecular signaling pathways such activity stimulates, is sufficient to induce neuronal survival and regeneration, particularly in retinal ganglion cells. Here, we review these data, discuss the effects of electrical activity on neurons' molecular signaling pathways and propose specific mechanisms by which exogenous electrical activity may be acting to enhance survival and regeneration.
View details for DOI 10.1088/1741-2560/65/055001
View details for Web of Science ID 000270670400003
View details for PubMedID 19721179
gamma-Synuclein as a marker of retinal ganglion cells
2008; 14 (182-84): 1540-1548
Previous studies have described gamma-synuclein as a protein highly expressed in retinal ganglion cells (RGCs), and a loss of RGCs correlates with a downregulation of gamma-synuclein gene expression in glaucoma. Here we asked whether gamma-synuclein expression in the retina can be considered a specific marker of RGCs.gamma-Synuclein expression was examined with immunohistochemistry in retinal sections from normal and glaucomatous human eyes. Primary cultures of RGCs from Sprague-Dawley rats purified by sequential immunopanning using a monoclonal antibody to Thy1-1, cultures of A7 immortalized optic nerve astrocytes from newborn rats, and the immortalized RGC-5 cell line were studied using immunofluorescence and quantitative RT-PCR.gamma-Synuclein was highly expressed in RGCs in the human retina and was localized in cytoplasm adjacent to the RGC nuclear marker, Brn-3a. Axons of RGCs were immunopositive for gamma-synuclein in the nerve fiber layer (NFL), the lamina cribrosa and the retrobulbar optic nerve. In the optic nerve of glaucoma patients, axon swellings were likewise immunopositive, whereas in the retina of patients with retinoblastoma, NFL staining appeared reduced. In primary rat RGCs and in immortalized RGC-5 cultures, gamma-synuclein was localized predominantly in the perinuclear area and in cell processes. Among rat retinal cells in culture, all Brn-3a positive cells were stained with a gamma-synuclein antibody; rare gamma-synuclein-positive cells were not stained by the Brn-3a antibody.gamma-Synuclein is selectively and abundantly expressed in human RGCs in vivo, primary rat RGCs in vitro, and immortalized RGC-5 cells. In pathology, gamma-synuclein abundance may vary between RGC somas and axons. Coincident Brn-3a and gamma-synuclein expression suggests that strong gamma-synuclein expression can be considered a marker of RGCs. Future translational approaches might include using a gamma-synuclein promoter for the specific delivery of siRNA or therapeutic proteins to RGCs.
View details for Web of Science ID 000258924200003
View details for PubMedID 18728752
View details for PubMedCentralID PMC2518532
A novel biological function for CD44 in axon growth of retinal ganglion cells identified by a bioinformatics approach
JOURNAL OF NEUROCHEMISTRY
2007; 103 (4): 1491-1505
The failure of CNS regeneration and subsequent motor and sensory loss remain major unsolved questions despite massive accumulation of experimental observations and results. The sheer volume of data and the variety of resources from which these data are generated make it difficult to integrate prior work to build new hypotheses. To address these challenges we developed a prototypic suite of computer programs to extract protein names from relevant publications and databases and associated each of them with several general categories of biological functions in nerve regeneration. To illustrate the usefulness of our data mining approach, we utilized the program output to generate a hypothesis for a biological function of CD44 interaction with osteopontin (OPN) and laminin in axon outgrowth of CNS neurons. We identified CD44 expression in retinal ganglion cells and when these neurons were plated on poly-l-lysine 3% of them initiated axon growth, on OPN 15%, on laminin-111 (1x) 41%, on laminin-111 (0.5x) 56%, and on a mixture of OPN and laminin (1x) 67% of neurons generated axon growth. With the aid of a deoxyribozyme (DNA enzyme) to CD44 that digests the target mRNA, we demonstrated that a reduction of CD44 expression led to reduced axon initiation of retinal ganglion cells on all substrates. We suggest that such an integrative, applied systems biology approach to CNS trauma will be critical to understand and ultimately overcome the failure of CNS regeneration.
View details for DOI 10.1111/j.1471-4159.2007.04858.x
View details for Web of Science ID 000250403500021
View details for PubMedID 17760872
View details for PubMedCentralID PMC2901540
Disease gene candidates revealed by expression profiling of retinal ganglion cell development
JOURNAL OF NEUROSCIENCE
2007; 27 (32): 8593-8603
To what extent do postmitotic neurons regulate gene expression during development or after injury? We took advantage of our ability to highly purify retinal ganglion cells (RGCs) to profile their pattern of gene expression at 13 ages from embryonic day 17 through postnatal day 21. We found that a large proportion of RGC genes are regulated dramatically throughout their postmitotic development, although the genes regulated through development in vivo generally are not regulated similarly by RGCs allowed to age in vitro. Interestingly, we found that genes regulated by developing RGCs are not generally correlated with genes regulated in RGCs stimulated to regenerate their axons. We unexpectedly found three genes associated with glaucoma, optineurin, cochlin, and CYP1B1 (cytochrome P450, family 1, subfamily B, polypeptide 1), previously thought to be primarily expressed in the trabecular meshwork, which are highly expressed by RGCs and regulated through their development. We also identified several other RGC genes that are encoded by loci linked to glaucoma. The expression of glaucoma-linked genes by RGCs suggests that, at least in some cases, RGCs may be directly involved in glaucoma pathogenesis rather than indirectly involved in response to increased intraocular pressure. Consistent with this hypothesis, we found that CYP1B1 overexpression potentiates RGC survival.
View details for DOI 10.1523/JNEUROSCI.4488-07.2007
View details for Web of Science ID 000248708400013
View details for PubMedID 17687037
View details for PubMedCentralID PMC2885852
Atypical mild enhanced S-Cone syndrome with novel compound heterozygosity of the NR2E3 gene
AMERICAN JOURNAL OF OPHTHALMOLOGY
2007; 144 (1): 157-159
To report mild enhanced s-cone syndrome (ESCS) associated with a novel heterozygous mutation of the NR2E3 gene.Observational case report.Clinical examination, optical coherence tomography (OCT), electroretinography (ERG), genetic analysis, and protein homology modeling.Examination of a 9-year-old girl with acute visual loss of the left eye showed visual acuity of 20/30 in the right eye and 20/200 in the left eye; OCT revealed a choroidal neovascular membrane (CNVM) in the left fovea and cystic maculopathy in the right eye. Full-field ERG showed supranormal s-cone responses, reduced rod response, and characteristic ESCS waveform in photopic cone response but not in scotopic bright-flash response. Sequence analysis revealed heterozygous mutations in the NR2E3 gene, c.767C-->T yielding a substitution p.Ala256Val, and a mutation in the splice site before exon 2, c.119-2 A-->C.The p.Ala256Val mutation affects the ligand binding domain of the NR2E3 nuclear receptor only, resulting in modestly impaired ESCS ERG results.
View details for Web of Science ID 000247867800038
View details for PubMedID 17601449
Eph-dependent tyrosine phosphorylation of ephexin1 modulates growth cone collapse
2005; 46 (2): 191-204
Ephs regulate growth cone repulsion, a process controlled by the actin cytoskeleton. The guanine nucleotide exchange factor (GEF) ephexin1 interacts with EphA4 and has been suggested to mediate the effect of EphA on the activity of Rho GTPases, key regulators of the cytoskeleton and axon guidance. Using cultured ephexin1-/- mouse neurons and RNA interference in the chick, we report that ephexin1 is required for normal axon outgrowth and ephrin-dependent axon repulsion. Ephexin1 becomes tyrosine phosphorylated in response to EphA signaling in neurons, and this phosphorylation event is required for growth cone collapse. Tyrosine phosphorylation of ephexin1 enhances ephexin1's GEF activity toward RhoA while not altering its activity toward Rac1 or Cdc42, thus changing the balance of GTPase activities. These findings reveal that ephexin1 plays a role in axon guidance and is regulated by a switch mechanism that is specifically tailored to control Eph-mediated growth cone collapse.
View details for DOI 10.1016/j.neuron.2005.01.030
View details for Web of Science ID 000228674800007
View details for PubMedID 15848799
Intrinsic neuronal regulation of axon and dendrite growth
CURRENT OPINION IN NEUROBIOLOGY
2004; 14 (5): 551-557
Neurons extend long axons and highly branched dendrites, and our understanding of the essential regulators of these processes has advanced in recent years. In the past year, investigators have shown that transcriptional control, posttranslational degradation and signaling cascades may be master regulators of axon and dendrite elongation and branching. Thus, evidence is mounting for the importance of the intrinsic growth state of a neuron as a crucial determinant of its ability to grow, or to regenerate, axons and dendrites.
View details for DOI 10.1016/j.conb.2004.08.012
View details for Web of Science ID 000224721200005
View details for PubMedID 15464887
Gene expression profiling of purified rat retinal ganglion cells
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE
2004; 45 (8): 2503-2513
The phenotype of specialized cells arises, in part, from their characteristic gene expression patterns. Retinal ganglion cells (RGCs) are of wide interest in neuroscience and die in glaucoma and other optic neuropathies. In this study the genes expressed by RGCs were profiled by expressed sequence tag (EST) analysis.ESTs were generated from a cDNA library constructed from RGCs isolated by immunopanning. The RGC genes were compared with published microarray expression profiles from 13 different neural regions. Immunohistochemistry was performed by standard methods.Clustering of 4791 RGC ESTs identified 2360 unique gene clusters. Of these, 60% represented known genes, 27% uncharacterized genes/ESTs, and 13% novel sequence. Unexpectedly, one of the largest RGC clusters, RESP18, corresponded to a neuroendocrine-specific gene preferentially expressed in the hypothalamus. RESP18 immunoreactivity within the retina was found mainly in the RGC layer. DDAH1, a gene involved in nitric oxide metabolism, was localized to RGC and amacrine layers. Comparison of gene expression patterns across neuronal regions revealed a prominent subset of RGC genes that were overexpressed in dorsal root and trigeminal ganglia. To narrow the search for candidate disease-related genes, RGC genes were mapped to known disease loci for optic neuropathies.This work is one of the first efforts to profile gene expression in a purified population of retinal neurons, the RGCs. The profiling, in addition to revealing both known and novel genes underlying the RGC phenotype, also uncovered common patterns of gene expression between RGCs and other sensory ganglia.
View details for DOI 10.1167/iovs.03-1391
View details for Web of Science ID 000222908500009
View details for PubMedID 15277470
An oligodendrocyte lineage-specific semaphorin, sema5A, inhibits axon growth by retinal ganglion cells
JOURNAL OF NEUROSCIENCE
2004; 24 (21): 4989-4999
In the mammalian CNS, glial cells repel axons during development and inhibit axon regeneration after injury. It is unknown whether the same repulsive axon guidance molecules expressed by glia and their precursors during development also play a role in inhibiting regeneration in the injured CNS. Here we investigate whether optic nerve glial cells express semaphorin family members and, if so, whether these semaphorins inhibit axon growth by retinal ganglion cells (RGCs). We show that each optic nerve glial cell type, astrocytes, oligodendrocytes, and their precursor cells, expressed a distinct complement of semaphorins. One of these, sema5A, was expressed only by purified oligodendrocytes and their precursors, but not by astrocytes, and was present in both normal and axotomized optic nerve but not in peripheral nerves. Sema5A induced collapse of RGC growth cones and inhibited RGC axon growth when presented as a substrate in vitro. To determine whether sema5A might contribute to inhibition of axon growth after injury, we studied the ability of RGCs to extend axons when cultured on postnatal day (P) 4, P8, and adult optic nerve explants and found that axon growth was strongly inhibited. Blocking sema5A using a neutralizing antibody significantly increased RGC axon growth on these optic nerve explants. These data support the hypothesis that sema5A expression by oligodendrocyte lineage cells contributes to the glial cues that inhibit CNS regeneration.
View details for DOI 10.1523/JNEUROSCI.4390-03.2004
View details for Web of Science ID 000221654400011
View details for PubMedID 15163691
Anaplasma phagocytophilum, Babesia microti, and Borrelia burgdorferi in Ixodes scapularis, southern coastal Maine
EMERGING INFECTIOUS DISEASES
2004; 10 (4): 744-746
Ixodes scapularis (deer ticks) from Maine were tested for multiple infections by polymerase chain reaction and immunofluorescence. In 1995, 29.5%, 9.5%, and 1.9% of deer ticks were infected with Borrelia burgdorferi, Anaplasma phagocytophilum, and Babesia microti, respectively. In 1996 and 1997, the number of A. phagocytophilum-infected ticks markedly declined. In 1995 through 1996, 4 (1.3%) of 301 were co-infected.
View details for Web of Science ID 000220578600034
View details for PubMedID 15200875
Gene expression profiling of purified retinal ganglion cells
Annual Meeting of the Association-for-Research-in-Vision-and-Ophthalmology
ASSOC RESEARCH VISION OPHTHALMOLOGY INC. 2003: U387–U387
View details for Web of Science ID 000184606802186
- How does an axon grow? GENES & DEVELOPMENT 2003; 17 (8): 941-958
Amacrine-signaled loss of intrinsic axon growth ability by retinal ganglion cells
2002; 296 (5574): 1860-1864
The central nervous system (CNS) loses the ability to regenerate early during development, but it is not known why. The retina has long served as a simple model system for study of CNS regeneration. Here we show that amacrine cells signal neonatal rat retinal ganglion cells (RGCs) to undergo a profound and apparently irreversible loss of intrinsic axon growth ability. Concurrently, retinal maturation triggers RGCs to greatly increase their dendritic growth ability. These results suggest that adult CNS neurons fail to regenerate not only because of CNS glial inhibition but also because of a loss of intrinsic axon growth ability.
View details for Web of Science ID 000176054300047
View details for PubMedID 12052959
Retinal ganglion cells do not extend axons by default: Promotion by neurotrophic signaling and electrical activity
2002; 33 (5): 689-702
We investigate the signaling mechanisms that induce retinal ganglion cell (RGC) axon elongation by asking whether surviving neurons extend axons by default. We show that bcl-2 overexpression is sufficient to keep purified RGCs alive in the absence of any glial or trophic support. The bcl-2-expressing RGCs do not extend axons or dendrites unless signaled to do so by single peptide trophic factors. Axon growth stimulated by peptide trophic factors is remarkably slow but is profoundly potentiated by physiological levels of electrical activity spontaneously generated within embryonic explants or mimicked on a multielectrode silicon chip. These findings demonstrate that these surviving neurons do not constitutively extend axons and provide insight into the signals that may be necessary to promote CNS regeneration.
View details for Web of Science ID 000174286200006
View details for PubMedID 11879647
EphA receptors regulate growth cone dynamics through the novel guanine nucleotide exchange factor ephexin
2001; 105 (2): 233-244
Eph receptors transduce short-range repulsive signals for axon guidance by modulating actin dynamics within growth cones. We report the cloning and characterization of ephexin, a novel Eph receptor-interacting protein that is a member of the Dbl family of guanine nucleotide exchange factors (GEFs) for Rho GTPases. Ephrin-A stimulation of EphA receptors modulates the activity of ephexin leading to RhoA activation, Cdc42 and Rac1 inhibition, and cell morphology changes. In addition, expression of a mutant form of ephexin in primary neurons interferes with ephrin-A-induced growth cone collapse. The association of ephexin with Eph receptors constitutes a molecular link between Eph receptors and the actin cytoskeleton and provides a novel mechanism for achieving highly localized regulation of growth cone motility.
View details for Web of Science ID 000168384300010
View details for PubMedID 11336673
The relationship between neuronal survival and regeneration
ANNUAL REVIEW OF NEUROSCIENCE
2000; 23: 579-612
The ability of peripheral nervous system (PNS) but not central nervous system (CNS) neurons to regenerate their axons is a striking peculiarity of higher vertebrates. Much research has focused on the inhibitory signals produced by CNS glia that thwart regenerating axons. Less attention has been paid to the injury-induced loss of trophic stimuli needed to promote the survival and regeneration of axotomized neurons. Could differences in the mechanisms that control CNS and PNS neuronal survival and growth also contribute to the disparity in regenerative capacity? Here we review recent studies concerning the nature of the signals necessary to promote neuronal survival and growth, with an emphasis on their significance to regeneration after CNS injury.
View details for Web of Science ID 000086730500020
View details for PubMedID 10845076
Neural regeneration: Extending axons from bench to brain
1998; 8 (9): R310-R312
Many studies have shown that myelin in the central nervous system strongly inhibits the regeneration of axons, so it comes as a surprise to discover that adult neurons transplanted into the brain rapidly extend their axons through myelinated pathways.
View details for Web of Science ID 000073343100011
View details for PubMedID 9560333