Kif5a Regulates Mitochondrial Transport in Developing Retinal Ganglion Cells In Vitro.
Investigative ophthalmology & visual science
2023; 64 (3): 4
Axon transport of organelles and neurotrophic factors is necessary for maintaining cellular function and survival of retinal ganglion cells (RGCs). However, it is not clear how trafficking of mitochondria, essential for RGC growth and maturation, changes during RGC development. The purpose of this study was to understand the dynamics and regulation of mitochondrial transport during RGC maturation using acutely purified RGCs as a model system.Primary RGCs were immunopanned from rats of either sex during three stages of development. MitoTracker dye and live-cell imaging were used to quantify mitochondrial motility. Analysis of single-cell RNA sequencing was used to identify Kinesin family member 5A (Kif5a) as a relevant motor candidate for mitochondrial transport. Kif5a expression was manipulated with either short hairpin RNA (shRNA) or exogenous expression adeno-associated virus viral vectors.Anterograde and retrograde mitochondrial trafficking and motility decreased through RGC development. Similarly, the expression of Kif5a, a motor protein that transports mitochondria, also decreased during development. Kif5a knockdown decreased anterograde mitochondrial transport, while Kif5a expression increased general mitochondrial motility and anterograde mitochondrial transport.Our results suggested that Kif5a directly regulates mitochondrial axonal transport in developing RGCs. Future work exploring the role of Kif5a in vivo in RGCs is indicated.
View details for DOI 10.1167/iovs.64.3.4
View details for PubMedID 36862119
Mettl14-mediated m6A modification ensures the cell-cycle progression of late-born retinal progenitor cells.
View details for DOI 10.1016/j.celrep.2023.112596
Directly induced human retinal ganglion cells mimic fetal RGCs and are neuroprotective after transplantation invivo.
Stem cell reports
Retinal ganglion cell (RGC) replacement therapy could restore vision in glaucoma and other optic neuropathies. We developed a rapid protocol for directly induced RGC (iRGC) differentiation from human stem cells, leveraging overexpression of NGN2. Neuronal morphology and neurite growth were observed within 1week of induction; characteristic RGC-specific gene expression confirmed identity. Calcium imaging demonstrated gamma-aminobutyric acid (GABA)-induced excitation characteristic of immature RGCs. Single-cell RNA sequencing showed more similarities between iRGCs and early-stage fetal human RGCs than retinal organoid-derived RGCs. Intravitreally transplanted iRGCs survived and migrated into host retinas independent of prior optic nerve trauma, but iRGCs protected host RGCs from neurodegeneration. These data demonstrate rapid iRGC generation invitro into an immature cell with high similarity to human fetal RGCs and capacity for retinal integration after transplantation and neuroprotective function after optic nerve injury. The simplicity of this system may benefit translational studies on human RGCs.
View details for DOI 10.1016/j.stemcr.2022.10.011
View details for PubMedID 36368332
Elk-1 regulates retinal ganglion cell axon regeneration after injury.
2022; 12 (1): 17446
Adult central nervous system (CNS) axons fail to regenerate after injury, and master regulators of the regenerative program remain to be identified. We analyzed the transcriptomes of retinal ganglion cells (RGCs) at 1 and 5days after optic nerve injury with and without a cocktail of strongly pro-regenerative factors to discover genes that regulate survival and regeneration. We used advanced bioinformatic analysis to identify the top transcriptional regulators of upstream genes and cross-referenced these with the regulators upstream of genes differentially expressed between embryonic RGCs that exhibit robust axon growth vs. postnatal RGCs where this potential has been lost. We established the transcriptional activator Elk-1 as the top regulator of RGC gene expression associated with axon outgrowth in both models. We demonstrate that Elk-1 is necessary and sufficient to promote RGC neuroprotection and regeneration in vivo, and is enhanced by manipulating specific phosphorylation sites. Finally, we co-manipulated Elk-1, PTEN, and REST, another transcription factor discovered in our analysis, and found Elk-1 to be downstream of PTEN and inhibited by REST in the survival and axon regenerative pathway in RGCs. These results uncover the basic mechanisms of regulation of survival and axon growth and reveal a novel, potent therapeutic strategy to promote neuroprotection and regeneration in the adult CNS.
View details for DOI 10.1038/s41598-022-21767-3
View details for PubMedID 36261683
Quantitative BONCAT (QBONCAT) allows identification of newly synthesized proteins after optic nerve injury.
The Journal of neuroscience : the official journal of the Society for Neuroscience
Retinal ganglion cells (RGCs) die after optic nerve trauma or in degenerative disease. However, acute changes in protein expression that may regulate RGC response to injury are not fully understood, and detailed methods to quantify new protein synthesis have not been tested. Here we develop and apply a new in vivo quantitative measure of newly synthesized proteins to examine changes occurring in the retina after optic nerve injury. Azidohomoalanine (AHA), a noncanonical amino acid, was injected intravitreally into the eyes of rodents of either sex with or without optic nerve injury. Isotope variants of biotin-alkyne were used for quantitative BONCAT (QBONCAT) mass spectrometry, allowing identification of protein synthesis and transport rate changes in over 1000 proteins at 1 or 5 days after optic nerve injury. In vitro screening showed several newly synthesized proteins regulate axon outgrowth in primary neurons in vitro This novel approach to targeted quantification of newly synthesized proteins after injury uncovers a dynamic translational response within broader proteostasis regulation and enhances our understanding of the cellular response to injury.Significance Statement:Optic nerve injury results in death and degeneration of retinal ganglion cells and their axons. The specific cellular response to injury, including changes in new protein synthesis, is obscured by existing proteins and protein degradation. In this study, we introduce QBONCAT to isolate and quantify acute protein synthesis and subsequent transport between cellular compartments. We identify novel candidate protein effectors of the regenerative response and uncover their regulation of axon growth in vitro, validating the utility of QBONCAT for the discovery of novel regulatory and therapeutic candidates after optic nerve injury.
View details for DOI 10.1523/JNEUROSCI.3100-20.2022
View details for PubMedID 35396330
Proteomic screen reveals diverse protein transport between connected neurons in the visual system.
1800; 38 (4): 110287
Intercellular transfer of toxic proteins between neurons is thought to contribute to neurodegenerative disease, but whether direct interneuronal protein transfer occurs in the healthy brain is not clear. To assess the prevalence and identity of transferred proteins and the cellular specificity of transfer, we biotinylated retinal ganglion cell proteins invivo and examined biotinylated proteins transported through the rodent visual circuit using microscopy, biochemistry, and mass spectrometry. Electron microscopy demonstrated preferential transfer of biotinylated proteins from retinogeniculate inputs to excitatory lateral geniculate nucleus (LGN) neurons compared with GABAergic neurons. An unbiased mass spectrometry-based screen identified 200 transneuronally transported proteins (TNTPs) isolated from the visual cortex. The majority of TNTPs are present in neuronal exosomes, and virally expressed TNTPs, including tau and beta-synuclein, were detected in isolated exosomes and postsynaptic neurons. Our data demonstrate transfer of diverse endogenous proteins between neurons in the healthy intact brain and suggest that TNTP transport may be mediated by exosomes.
View details for DOI 10.1016/j.celrep.2021.110287
View details for PubMedID 35081342
Quantitative transportomics identifies Kif5a as a major regulator of neurodegeneration.
Many neurons in the adult central nervous system, including retinal ganglion cells (RGCs), degenerate and die after injury. Early axon protein and organelle trafficking failure is a key component in many neurodegenerative disorders yet changes to axoplasmic transport in disease models have not been quantified. We analyzed early changes in the protein 'transportome' from (RGC somas to their axons after optic nerve injury and identified transport failure of an anterograde motor protein Kif5a early in RGC degeneration. We demonstrated that manipulating Kif5a expression affects anterograde mitochondrial trafficking in RGCs and characterized axon transport in Kif5a knockout mice to identify proteins whose axon localization was Kif5a-dependent. Finally, we found that knockout of Kif5a in RGCs resulted in progressive RGC degeneration in the absence of injury. Together with expression data localizing Kif5a to human RGCs, these data identify Kif5a transport failure as a cause of RGC neurodegeneration and point to a mechanism for future therapeutics.
View details for DOI 10.7554/eLife.68148
View details for PubMedID 35259089
Assessment of Respiratory Droplet Transmission During the Ophthalmic Slit-Lamp Exam: A Particle Tracking Analysis
AMERICAN JOURNAL OF OPHTHALMOLOGY
2021; 222: 76-81
The global COVID-19 pandemic has resulted in a renewed focus on the importance of personal protective equipment (PPE) and other interventions to decrease spread of infectious diseases. Although several ophthalmology organizations have released guidance on appropriate PPE for surgical procedures and ophthalmology clinics, there is limited experimental evidence that demonstrates the efficacy of various interventions that have been suggested. In this study, we evaluated high-risk aspects of the slit-lamp exam and the effect of various PPE interventions, specifically the use of a surgical mask and a slit-lamp shield.Experimental simulation study.This was a single-center study in a patient simulation population. This study examined the presence of particles in the air near or on a slit-lamp, a simulated slit-lamp examiner, or a simulated patient using a fluorescent surrogate of respiratory droplets.Simulated coughing without a mask or slit-lamp shield resulted in widespread dispersion of fluorescent droplets during the model slit-lamp examination. Coughing with a mask resulted in the most significant decrease in droplets; however, particles still escaped from the top of the mask. Coughing with the slit-lamp shield alone blocked most of forward particle dispersion; however, significant distributions of respiratory droplets were found on the slit-lamp joystick and table. Coughing with both a mask and slit-lamp shield resulted in the least dispersion to the simulated examiner and the simulated patient. Scanning electron microscopy demonstrated particle sizes of 3-100 μm.Masking had the greatest effect in limiting spread of respiratory droplets, whereas slit-lamp shields and gloves also contributed to limiting exposure to droplets from SARS-CoV-2 during slit-lamp examination.
View details for DOI 10.1016/j.ajo.2020.08.046
View details for Web of Science ID 000625478500010
View details for PubMedID 32980331
View details for PubMedCentralID PMC7516395
Assessing the Physiological Relevance of Cough Simulators for Respiratory Droplet Dispersion
JOURNAL OF CLINICAL MEDICINE
2020; 9 (9)
Various breathing and cough simulators have been used to model respiratory droplet dispersion and viral droplets, in particular for SARS-CoV-2 modeling. However, limited data are available comparing these cough simulations to physiological breathing and coughing. In this study, three different cough simulators (Teleflex Mucosal Atomization Device Nasal (MAD Nasal), a spray gun, and GloGermTM MIST) that have been used in the literature were studied to assess their physiologic relevance. Droplet size, velocity, dispersion, and force generated by the simulators were measured. Droplet size was measured with scanning electron microscopy (SEM). Slow-motion videography was used to 3D reconstruct and measure the velocity of each simulated cough. A force-sensitive resistor was used to measure the force of each simulated cough. The average size of droplets from each cough simulator was 176 to 220 µm. MAD Nasal, the spray gun, and GloGermTM MIST traveled 0.38 m, 0.89 m, and 1.62 m respectively. The average velocities for the MAD Nasal, spray gun, and GloGermTM MIST were 1.57 m/s, 2.60 m/s, and 9.27 m/s respectively, and all yielded a force of <0.5 Newtons. GloGermTM MIST and the spray gun most closely resemble physiological coughs and breathing respectively. In conclusion, none of the simulators tested accurately modeled all physiologic characteristics (droplet size, 3-D dispersion velocity, and force) of a cough, while there were various strengths and weaknesses of each method. One should take this into account when performing simulations with these devices.
View details for DOI 10.3390/jcm9093002
View details for Web of Science ID 000581216200001
View details for PubMedID 32957639
View details for PubMedCentralID PMC7564804
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
Cholinergic neural activity directs retinal layer-specific angiogenesis and blood retinal barrier formation
2019; 10: 2477
Blood vessels in the central nervous system (CNS) develop unique features, but the contribution of CNS neurons to regulating those features is not fully understood. We report that inhibiting spontaneous cholinergic activity or reducing starburst amacrine cell numbers prevents invasion of endothelial cells into the deep layers of the retina and causes blood-retinal-barrier (BRB) dysfunction in mice. Vascular endothelial growth factor (VEGF), which drives angiogenesis, and Norrin, a Wnt ligand that induces BRB properties, are decreased after activity blockade. Exogenous VEGF restores vessel growth but not BRB function, whereas stabilizing beta-catenin in endothelial cells rescues BRB dysfunction but not vessel formation. We further identify that inhibiting cholinergic activity reduces angiogenesis during oxygen-induced retinopathy. Our findings demonstrate that neural activity lies upstream of VEGF and Norrin, coordinating angiogenesis and BRB formation. Neural activity originating from specific neural circuits may be a general mechanism for driving regional angiogenesis and barrier formation across CNS development.
View details for DOI 10.1038/s41467-019-10219-8
View details for Web of Science ID 000470248100009
View details for PubMedID 31171770
View details for PubMedCentralID PMC6554348
- The Role of Axon Transport in Neuroprotection and Regeneration DEVELOPMENTAL NEUROBIOLOGY 2018; 78 (10): 998-1010
Mass Spectrometry-Based Visualization of Molecules Associated with Human Habitats
2016; 88 (22): 10775-10784
The cars we drive, the homes we live in, the restaurants we visit, and the laboratories and offices we work in are all a part of the modern human habitat. Remarkably, little is known about the diversity of chemicals present in these environments and to what degree molecules from our bodies influence the built environment that surrounds us and vice versa. We therefore set out to visualize the chemical diversity of five built human habitats together with their occupants, to provide a snapshot of the various molecules to which humans are exposed on a daily basis. The molecular inventory was obtained through untargeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of samples from each human habitat and from the people that occupy those habitats. Mapping MS-derived data onto 3D models of the environments showed that frequently touched surfaces, such as handles (e.g., door, bicycle), resemble the molecular fingerprint of the human skin more closely than other surfaces that are less frequently in direct contact with humans (e.g., wall, bicycle frame). Approximately 50% of the MS/MS spectra detected were shared between people and the environment. Personal care products, plasticizers, cleaning supplies, food, food additives, and even medications that were found to be a part of the human habitat. The annotations indicate that significant transfer of chemicals takes place between us and our built environment. The workflows applied here will lay the foundation for future studies of molecular distributions in medical, forensic, architectural, space exploration, and environmental applications.
View details for DOI 10.1021/acs.analchem.6b03456
View details for Web of Science ID 000388154700001
View details for PubMedID 27732780
Maps of interaural delay in the owl's nucleus laminaris
JOURNAL OF NEUROPHYSIOLOGY
2015; 114 (3): 1862-1873
Axons from the nucleus magnocellularis form a presynaptic map of interaural time differences (ITDs) in the nucleus laminaris (NL). These inputs generate a field potential that varies systematically with recording position and can be used to measure the map of ITDs. In the barn owl, the representation of best ITD shifts with mediolateral position in NL, so as to form continuous, smoothly overlapping maps of ITD with iso-ITD contours that are not parallel to the NL border. Frontal space (0°) is, however, represented throughout and thus overrepresented with respect to the periphery. Measurements of presynaptic conduction delay, combined with a model of delay line conduction velocity, reveal that conduction delays can account for the mediolateral shifts in the map of ITD.
View details for DOI 10.1152/jn.00644.2015
View details for Web of Science ID 000362045100046
View details for PubMedID 26224776
View details for PubMedCentralID PMC4575979
A functional circuit model of interaural time difference processing
JOURNAL OF NEUROPHYSIOLOGY
2014; 112 (11): 2850-2864
Inputs from the two sides of the brain interact to create maps of interaural time difference (ITD) in the nucleus laminaris of birds. How inputs from each side are matched with high temporal precision in ITD-sensitive circuits is unknown, given the differences in input path lengths from each side. To understand this problem in birds, we modeled the geometry of the input axons and their corresponding conduction velocities and latencies. Consistent with existing physiological data, we assumed a common latency up to the border of nucleus laminaris. We analyzed two biological implementations of the model, the single ITD map in chickens and the multiple maps of ITD in barn owls. For binaural inputs, since ipsi- and contralateral initial common latencies were very similar, we could restrict adaptive regulation of conduction velocity to within the nucleus. Other model applications include the simultaneous derivation of multiple conduction velocities from one set of measurements and the demonstration that contours with the same ITD cannot be parallel to the border of nucleus laminaris in the owl. Physiological tests of the predictions of the model demonstrate its validity and robustness. This model may have relevance not only for auditory processing but also for other computational tasks that require adaptive regulation of conduction velocity.
View details for DOI 10.1152/jn.00484.2014
View details for Web of Science ID 000346023000016
View details for PubMedID 25185809
View details for PubMedCentralID PMC4254871
Maps of ITD in the Nucleus Laminaris of the Barn Owl
SPRINGER. 2013: 215-222
Axons from the nucleus magnocellularis (NM) and their targets in nucleus laminaris (NL) form the circuit responsible for encoding interaural time differences (ITDs). In barn owls, NL receives bilateral inputs from NM such that axons from the ipsilateral NM enter NL dorsally, while contralateral axons enter from the ventral side. These afferents and their synapses on NL neurons generate a tone-induced local field potential, or neurophonic, that varies systematically with position in NL. From dorsal to ventral within the nucleus, the best interaural time difference (ITD) of the neurophonic shifts from contralateral space to best ITDs around 0 µs. Earlier recordings suggested that in NL, iso-delay contours ran parallel to the dorsal and ventral borders of NL (Sullivan WE, Konishi M. Proc Natl Acad Sci U S A 83:8400-8404, 1986). This axis is orthogonal to that seen in chicken NL, where a single map of ITD runs from around 0 µs ITD medially to contralateral space laterally (Köppl C, Carr CE. Biol Cyber 98:541-559, 2008). Yet the trajectories of the NM axons are similar in owl and chicken (Seidl AH, Rubel EW, Harris DM, J Neurosci 30:70-80, 2010). We therefore used clicks to measure conduction time in NL and made lesions to mark the 0 µs iso-delay contour in multiple penetrations along an isofrequency slab. Iso-delay contours were not parallel to the dorsal and ventral borders of NL; instead the 0 µs iso-delay contour shifted systematically from a dorsal position in medial NL to a ventral position in lateral NL. Could different conduction delays account for the mediolateral shift in the representation of 0 µs ITD? We measured conduction delays using the neurophonic potential and developed a simple linear model of the delay-line conduction velocity. We then raised young owls with time-delaying earplugs in one ear (Gold JI, Knudsen EI, J Neurophysiol 82:2197-2209, 1999) to examine map plasticity.
View details for DOI 10.1007/978-1-4614-1590-9_24
View details for Web of Science ID 000333329600025
View details for PubMedID 23716226
View details for PubMedCentralID PMC4922490
Twice-weekly fluconazole prophylaxis in premature infants: Association with cholestasis
2011; 53 (4): 475-479
Fluconazole prophylaxis is effective in preventing invasive candidiasis in extremely low-birthweight (ELBW) infants. The authors previously reported an increased incidence of cholestasis with fluconazole prophylaxis in ELBW infants, which led to fluconazole prophylaxis being changed to a less frequent dosing (LFD) schedule of twice a week at their institution. The purpose of the present study was therefore to evaluate the effectiveness and safety of LFD fluconazole prophylaxis in preventing invasive candidiasis in ELBW infants.ELBW infants who received the LFD regimen of fluconazole (twice a week for up to 6 weeks) were compared with infants who received the frequent dosing (FD) schedule (every 72 h for first 2 weeks, every 48 h for next 2 weeks and every 24 h for the final 2 weeks). The two groups were compared for baseline demographics, risk factors for candidiasis, the rate of invasive fungal infection and the incidence and severity of cholestasis.There was no significant difference in the incidence of invasive candidiasis in infants who received the LFD (2/104, 2%) compared to FD (0/140, 0%; P= 0.4) fluconazole prophylaxis. The severity of cholestasis was lower and a trend towards decreased incidence of cholestasis was observed on the LFD schedule.The LFD regimen of fluconazole prophylaxis is effective in preventing invasive fungal infection in ELBW infants. The severity of cholestasis was decreased with the LFD schedule.
View details for DOI 10.1111/j.1442-200X.2010.03286.x
View details for Web of Science ID 000294026800009
View details for PubMedID 21040197
The Effect of Comprehensive Infection Control Measures on the Rate of Late-Onset Bloodstream Infections in Very Low-Birth-Weight Infants
AMERICAN JOURNAL OF PERINATOLOGY
2011; 28 (3): 227-232
Late-onset bloodstream infection (LOBI) is a significant problem in very low-birth-weight (VLBW) infants and can lead to increased mortality and morbidity. The incidence of LOBI in VLBW infants in our unit was >35% before 2004, much higher than 20% reported in other studies. A comprehensive infection control measure was introduced in our unit in 2005. Here we report the effects of comprehensive infection control measures on the rate of LOBI in VLBW infants. Infants in the preintervention group (born 2001 to 2004) were compared with the intervention group (born 2005 to 2008) for baseline demographics, risk factors for infection, and the rate of LOBI. LOBI was defined as a positive blood and/or cerebrospinal fluid culture after 3 days of life. Three hundred thirty-four VLBW infants were admitted to our unit during the preintervention period and 303 during the intervention period. There was no significant difference in baseline demographics and risk factors for LOBI between the two groups. The incidence of LOBI was significantly reduced from 38% before intervention to 23% after intervention ( P < 0.001). Comprehensive infection control measures significantly reduced the rate of LOBI in VLBW infants.
View details for DOI 10.1055/s-0030-1268237
View details for Web of Science ID 000287962000009
View details for PubMedID 20981639