The C-Terminus and Third Cytoplasmic Loop Cooperatively Activate Mouse Melanopsin Phototransduction
2020; 119 (2): 389?401
Optic Nerve Crush in Mice to Study Retinal Ganglion Cell Survival and Regeneration.
2020; 10 (6)
Melanopsin, an atypical vertebrate visual pigment, mediates non-image-forming light responses including circadian photoentrainment and pupillary light reflexes and contrast detection for image formation. Melanopsin-expressing intrinsically photosensitive retinal ganglion cells are characterized by sluggish activation and deactivation of their light responses. The molecular determinants of mouse melanopsin's deactivation have been characterized (i.e., C-terminal phosphorylation and ?-arrestin binding), but a detailed analysis of melanopsin's activation is lacking. We propose that an extended third cytoplasmic loop is adjacent to the proximal C-terminal region of mouse melanopsin in the inactive conformation, which is stabilized by the ionic interaction of these two regions. This model is supported by site-directed spin labeling and electron paramagnetic resonance spectroscopy of melanopsin, the results of which suggests a high degree of steric freedom at the third cytoplasmic loop, which is increased upon C-terminus truncation, supporting the idea that these two regions are close in three-dimensional space in wild-type melanopsin. To test for a functionally critical C-terminal conformation, calcium imaging of melanopsin mutants including a proximal C-terminus truncation (at residue 365) and proline mutation of this proximal region (H377P, L380P, Y382P) delayed melanopsin's activation rate. Mutation of all potential phosphorylation sites, including a highly conserved tyrosine residue (Y382), into alanines also delayed the activation rate. A comparison of mouse melanopsin with armadillo melanopsin-which has substitutions of various potential phosphorylation sites and a substitution of the conserved tyrosine-indicates that substitution of these potential phosphorylation sites and the tyrosine residue result in dramatically slower activation kinetics, a finding that also supports the role of phosphorylation in signaling activation. We therefore propose that melanopsin's C-terminus is proximal to intracellular loop 3, and C-terminal phosphorylation permits the ionic interaction between these two regions, thus forming a stable structural conformation that is critical for initiating G-protein signaling.
View details for DOI 10.1016/j.bpj.2020.06.013
View details for Web of Science ID 000552027100016
View details for PubMedID 32621866
View details for PubMedCentralID PMC7376183
MEF2 transcription factors differentially contribute to retinal ganglion cell loss after optic nerve injury.
2020; 15 (12): e0242884
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
Physiologic maturation is both extrinsically and intrinsically regulated in progenitor-derived neurons.
2020; 10 (1): 2337
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
Opposing Effects of Growth and Differentiation Factors in Cell-Fate Specification.
Current biology : CB
During development, newly-differentiated neurons undergo several morphological and physiological changes to become functional, mature neurons. Physiologic maturation of neuronal cells derived from isolated stem or progenitor cells may provide insight into maturation in vivo but is not well studied. As a step towards understanding how neuronal maturation is regulated, we studied the developmental switch of response to the neurotransmitter GABA, from excitatory depolarization to inhibitory hyperpolarization. We compared acutely isolated retinal ganglion cells (RGCs) at various developmental stages and RGCs differentiated in vitro from embryonic retinal progenitors for the effects of aging and, independently, of retinal environment age on their GABAA receptor (GABAAR) responses, elicited by muscimol. We found that neurons generated in vitro from progenitors exhibited depolarizing, immature GABA responses, like those of early postnatal RGCs. As progenitor-derived neurons aged from 1 to 3 weeks, their GABA responses matured. Interestingly, signals secreted by the early postnatal retina suppressed acquisition of mature GABA responses. This suppression was not associated with changes in expression of GABAAR or of the chloride co-transporter KCC2, but rather with inhibition of KCC2 dimerization in differentiating neurons. Taken together, these data indicate GABA response maturation depends on release of inhibition by developmentally regulated diffusible signals from the retina.
View details for DOI 10.1038/s41598-020-58120-5
View details for PubMedID 32047174
Regulation of Neuronal Survival and Axon Growth by a Perinuclear cAMP Compartment.
The Journal of neuroscience : the official journal of the Society for Neuroscience
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-?) 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
Intracellular compartmentation of cAMP promotes neuroprotection and regeneration of CNS neurons.
Neural regeneration research
2017; 12 (2): 201?2
NEUROREGENERATION. Promoting CNS repair.
2016; 353 (6294): 30-31
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
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 mAKAP? promotes neuronal survival and axon growth. mAKAP? 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 mAKAP?-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 mAKAP? 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
beta-Arrestin-Dependent Deactivation of Mouse Melanopsin
2014; 9 (11)
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-? (mAKAP?) is required for the survival and axon growth of cultured primary RGCs. Although genetic deletion of mAKAP? 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 mAKAP? in the adult increased RGC death after optic nerve crush. Importantly, mAKAP? was required for the neuroprotective effects of brain-derived neurotrophic factor and cyclic adenosine-monophosphate (cAMP) after injury. These results identify mAKAP? 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
Characterization of visual pigments, oil droplets, lens and cornea in the whooping crane Grus americana
JOURNAL OF EXPERIMENTAL BIOLOGY
2014; 217 (21): 3883-3890
In mammals, the expression of the unusual visual pigment, melanopsin, is restricted to a small subset of intrinsically photosensitive retinal ganglion cells (ipRGCs), whose signaling regulate numerous non-visual functions including sleep, circadian photoentrainment and pupillary constriction. IpRGCs exhibit attenuated electrical responses following sequential and prolonged light exposures indicative of an adaptational response. The molecular mechanisms underlying deactivation and adaptation in ipRGCs however, have yet to be fully elucidated. The role of melanopsin phosphorylation and ?-arrestin binding in this adaptive process is suggested by the phosphorylation-dependent reduction of melanopsin signaling in vitro and the ubiquitous expression of ?-arrestin in the retina. These observations, along with the conspicuous absence of visual arrestin in ipRGCs, suggest that a ?-arrestin terminates melanopsin signaling. Here, we describe a light- and phosphorylation- dependent reduction in melanopsin signaling mediated by both ?-arrestin 1 and ?-arrestin 2. Using an in vitro calcium imaging assay, we demonstrate that increasing the cellular concentration of ?-arrestin 1 and ?-arrestin 2 significantly increases the rate of deactivation of light-activated melanopsin in HEK293 cells. Furthermore, we show that this response is dependent on melanopsin carboxyl-tail phosphorylation. Crosslinking and co-immunoprecipitation experiments confirm ?-arrestin 1 and ?-arrestin 2 bind to melanopsin in a light- and phosphorylation- dependent manner. These data are further supported by proximity ligation assays (PLA), which demonstrate a melanopsin/?-arrestin interaction in HEK293 cells and ipRGCs. Together, these results suggest that melanopsin signaling is terminated in a light- and phosphorylation-dependent manner through the binding of a ?-arrestin within the retina.
View details for DOI 10.1371/journal.pone.0113138
View details for Web of Science ID 000345158700116
View details for PubMedID 25401926
Identification of Critical Phosphorylation Sites on the Carboxy Tail of Melanopsin
2014; 53 (16): 2644-2649
Vision has been investigated in many species of birds, but few studies have considered the visual systems of large birds and the particular implications of large eyes and long-life spans on visual system capabilities. To address these issues we investigated the visual system of the whooping crane Grus americana (Gruiformes, Gruidae), which is one of only two North American crane species. It is a large, long-lived bird in which UV sensitivity might be reduced by chromatic aberration and entrance of UV radiation into the eye could be detrimental to retinal tissues. To investigate the whooping crane visual system we used microspectrophotometry to determine the absorbance spectra of retinal oil droplets and to investigate whether the ocular media (i.e. the lens and cornea) absorb UV radiation. In vitro expression and reconstitution was used to determine the absorbance spectra of rod and cone visual pigments. The rod visual pigments had wavelengths of peak absorbance (?max) at 500 nm, whereas the cone visual pigment ?max values were determined to be 404 nm (SWS1), 450 nm (SWS2), 499 nm (RH2) and 561 nm (LWS), similar to other characterized bird visual pigment absorbance values. The oil droplet cut-off wavelength (?cut) values similarly fell within ranges recorded in other avian species: 576 nm (R-type), 522 nm (Y-type), 506 nm (P-type) and 448 nm (C-type). We confirm that G. americana has a violet-sensitive visual system; however, as a consequence of the ?max of the SWS1 visual pigment (404 nm), it might also have some UV sensitivity.
View details for DOI 10.1242/jeb.108456
View details for Web of Science ID 000344866300022
View details for PubMedID 25267845
Shedding new light on opsin evolution
PROCEEDINGS OF THE ROYAL SOCIETY B-BIOLOGICAL SCIENCES
2012; 279 (1726): 3-14
Light-activated opsins undergo carboxy-terminal phosphorylation, which contributes to the deactivation of their photoresponse. The photopigment melanopsin possesses an unusually long carboxy tail containing 37 serine and threonine sites that are potential sites for phosphorylation by a G-protein dependent kinase (GRK). Here, we show that a small cluster of six to seven sites is sufficient for deactivation of light-activated mouse melanopsin. Surprisingly, these sites are distinct from those that regulate deactivation of rhodopsin. In zebrafish, there are five different melanopsin genes that encode proteins with distinct carboxy-terminal domains. Naturally occurring changes in the same cluster of phosphorylatable amino acids provides diversity in the deactivation kinetics of the zebrafish proteins. These results suggest that variation in phosphorylation sites provides flexibility in the duration and kinetics of melanopsin-mediated light responses.
View details for DOI 10.1021/bi401724r
View details for Web of Science ID 000335297200009
View details for PubMedID 24678795
Opsin proteins are essential molecules in mediating the ability of animals to detect and use light for diverse biological functions. Therefore, understanding the evolutionary history of opsins is key to understanding the evolution of light detection and photoreception in animals. As genomic data have appeared and rapidly expanded in quantity, it has become possible to analyse opsins that functionally and histologically are less well characterized, and thus to examine opsin evolution strictly from a genetic perspective. We have incorporated these new data into a large-scale, genome-based analysis of opsin evolution. We use an extensive phylogeny of currently known opsin sequence diversity as a foundation for examining the evolutionary distributions of key functional features within the opsin clade. This new analysis illustrates the lability of opsin protein-expression patterns, site-specific functionality (i.e. counterion position) and G-protein binding interactions. Further, it demonstrates the limitations of current model organisms, and highlights the need for further characterization of many of the opsin sequence groups with unknown function.
View details for DOI 10.1098/rspb.2011.1819
View details for Web of Science ID 000297674300002
View details for PubMedID 22012981