Evan G. Cameron

Shedding new light on opsin evolution.

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.

Identification of critical phosphorylation sites on the carboxy tail of melanopsin

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.

β-Arrestin-dependent deactivation of mouse melanopsin.

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 β-arrestin binding in this adaptive process is suggested by the phosphorylation-dependent reduction of melanopsin signaling in vitro and the ubiquitous expression of β-arrestin in the retina. These observations, along with the conspicuous absence of visual arrestin in ipRGCs, suggest that a β-arrestin terminates melanopsin signaling. Here, we describe a light- and phosphorylation- dependent reduction in melanopsin signaling mediated by both β-arrestin 1 and β-arrestin 2. Using an in vitro calcium imaging assay, we demonstrate that increasing the cellular concentration of β-arrestin 1 and β-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 β-arrestin 1 and β-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/β-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 β-arrestin within the retina.

Characterization of visual pigments, oil droplets, lens and cornea in the whooping crane Grus americana

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 (λmax) at 500 nm, whereas the cone visual pigment λmax 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 (λcut) 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 λmax of the SWS1 visual pigment (404 nm), it might also have some UV sensitivity.

Muscle A-Kinase Anchoring Protein-α is an Injury-Specific Signaling Scaffold Required for Neurotrophic- and Cyclic Adenosine Monophosphate-Mediated Survival

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.

Intracellular compartmentation of cAMP promotes neuroprotection and regeneration of CNS neurons

In the central nervous system (CNS), cyclic adenosine monophosphate (cAMP) plays a critical role in numerous, often concurrent, neuronal functions including survival, growth, differentiation and synaptogenesis. Elucidating the mechanisms by which this ubiquitous secondary messenger influences these processes is crucial to understanding why CNS neurons fail to regenerate after injury or in disease. Several survival and growth promoting pathways have been linked to cAMP signaling in neurons, including mitogen-activated protein kinase (MAPK), phosphatase and tensin homolog (PTEN), and signal transducer and activator of transcription 3 (STAT3), however, the molecular mechanisms by which cAMP specifically influences these pathways is still unclear. Recent evidence suggests that the context, extent and means by which cAMP signaling takes place account for its ability to simultaneously regulate survival and growth within neurons (Corredor et al., 2012; Wang and Cameron et al., 2015; Averaimo et al., 2016). These findings raise further questions about how cAMP signaling and survival signaling itself change after injury or in disease, and to what degree the intracellular compartmentation of cAMP signals is critical for its function in regulating neuroprotection and regeneration (Cameron and Goldberg, 2016). In mammals, cAMP is synthesized by a family of nine transmembrane adenylyl cyclases (tmACs, AC1-9) and one bicarbonate-and calcium-sensitive soluble adenylyl cyclase (sAC, AC10). Neurons express six tmACs, as well as sAC, and thus likely employ multiple cAMP signaling mechanisms to regulate survival and growth. In retinal ganglion cell (RGC) neurons, electrical activity potentiates neurotrophic responsiveness in a cAMP-dependent manner, presumably through the increase of intracellular calcium. Interestingly, pharmacological inhibition and gene deletion of calcium-sensitive tmACs have no effect on baseline RGC survival or growth (Corredor et al., 2012). Conversely, inhibiting sAC signaling decreases RGC growth, and sAC gene knockout (KO) severely perturbs normal RGC and photoreceptor differentiation (Shaw et al., 2016). Thus, it appears that sAC, and not tmACs, is the major source of cAMP that drives survival and growth signaling in CNS neurons. Still, these findings only partially explain how sAC-derived cAMP may influence these processes, as sAC is expressed in multiple subcellular compartments including the nucleus, mitochondria and cytoplasm (Corredor et al., 2012). Further still, cAMP activates three distinct effectors, protein kinase A (PKA), exchange protein directly activated by cAMP (EPACs) and cyclic nucleotide-gated channels (CNGC), which themselves can differentially influence numerous downstream signaling pathways. How then does sAC-derived cAMP precisely modulate survival and growth in CNS neurons? A general hypothesis is that cAMP specificity is achieved through compartmentalization and local control of cAMP dynamics. This notion is supported by a report that ephrin-dependent retraction of axonal arbors was reliant on cAMP signaling around lipid rafts (Averaimo et al., 2016). Moreover, axon retraction could only be provoked by acute changes in cAMP and not sustained cAMP elevation, underscoring the multiple ways in which cAMP must be precisely controlled to elicit a specific effect. In addition to regulated and compartmentalized cAMP synthesis, cAMP dynamics are modulated by phosphodiesterases (PDEs), a large, diverse family of enzymes that degrade cyclic nucleotides and control their concentration, localization and lifetime. PDE activity has been linked to neuronal survival, growth and synapse formation. For example, Rolipram, a selective PDE4 inhibitor, reportedly promotes neuroprotection and perturbs axon dieback following acute spinal cord injury (Schaal et al., 2012). Further, synaptic plasticity and memory can be impaired by expression of a full length PDE4A5 isozyme in hippocampal neurons that associates with dendritic compartments but not a PDE4A5 truncation mutant (lacking an N-terminal targeting domain) that localizes exclusively in the perinuclear space (Havekes et al., 2016). Together, these data support the hypothesis that subcellular localization and strict control of cAMP levels by sAC and PDEs impart the specificity required to regulate growth, survival and synaptogenesis in CNS neurons. But what dictates where sAC-derived cAMP signaling takes place within a neuron? Spatial-temporal control of cAMP signaling is often conferred by a heterogeneous family of multivalent scaffold proteins called A-kinase anchoring proteins (AKAP), so-called due to their binding of the cAMP effector PKA. AKAPs are expressed in every cell type, but have been especially well-characterized in cardiac myocytes and neurons where they facilitate localized signaling within distinct microdomains through the organization of signalosomes containing specific isoforms of ACs, PDEs and PKAs. In addition, AKAPs enable crosstalk between the cAMP pathway and known regulators of neuronal growth and survival such as extracellular signal-regulated kinases (ERKs) and calcium-regulated nuclear factor of activated T-cells (NFAT) tran-

Promoting CNS repair

What influences glial and neuronal response to neurodegeneration? A developmental loss of intrinsic reparative capacity and the inhibitory environment in injury and disease contribute to regenerative failure in the central nervous system (CNS). The same factors are thought to hinder endogenous and exogenous regenerative therapies, including cell-based replacement (1, 2). In neurodegenerative disorders, the contributions of microglia, astrocytes, and peripheral immune cells may be both harmful and beneficial. For example, resident microglia and peripheral cells of the innate immune system promote inflammation and cell death (apoptosis) in response to CNS injury, but immune cell activation also has been associated with neuroprotection and repair (3). This duality suggests that stimulating protective functions while minimizing proapoptotic and inhibitory signals could prove critical in treating neurodegenerative disease. On page 43 of this issue, Neves et al. (4) show that a neurotrophic signaling pathway in microglia and innate immune cells that is activated in disease or injury can be leveraged to promote neuroprotection and tissue repair.

A Stochastic Model of the Melanopsin Phototransduction Cascade

Melanopsin is an unusual vertebrate photopigment that, in mammals, is expressed in a small subset of intrinsically photosensitive retinal ganglion cells (ipRGCs), whose signaling has been implicated in non-image forming vision, regulating such functions as circadian rhythms, pupillary light reflex, and sleep. The biochemical cascade underlying the light response in ipRGCs has not yet been fully elucidated. We developed a stochastic model of the hypothesized melanopsin phototransduction cascade and illustrated that the stochastic model can qualitatively reproduce experimental results under several different conditions. The model allows us to probe various mechanisms in the phototransduction cascade in a way that is not currently experimentally feasible.

Determining the Role of Melanopsin C-Tail in Deactivation and Trafficking

Melanopsin is a unique non-image forming visual pigment expressed in intrinsically photosensitive retinal ganglion cells in the vertebrate retina. These cells are involved in many non-image forming functions such as the photoentrainment of circadian rhythm and the pupillary light reflex. Melanopsin is deactivated through the phosphorylation of the C-tail followed by the binding of a β-arrestin molecule. β-arrestin contains a signal on its C-terminus that allows for internalization of G-protein coupled receptors (GPCRs) after inactivation. However, it is currently unknown whether melanopsin is internalized. Angiotensin II type 1A receptor (ATII1AR) and B2 adrenergic receptor (B2AR) are two GPCRs known to bind β-arrestin and undergo endocytosis. To study the role of the C-tail in melanopsin deactivation and trafficking, the C-tail of melanopsin is replaced with either ATII1AR or B2AR c-tail using cloning techniques. We then introduce our plasmids into Human Embryonic Kidney (HEK) cells to assess the localization and signaling of the constructs. Sequencing has confirmed that several chimeric constructs have successfully been made. Calcium imaging has confirmed that the Mel/B2AR chimeric constructs signals in a similar manner as melanopsin in the presence of light. These results will help determine the role of the melanopsin C-tail in its deactivation and trafficking.