Abstract Inherited retinal degenerations originate from mutations in >300 genes, many of which cause the production of misfolded mutant photoreceptor proteins that are ultimately degraded by the ubiquitin-proteasome system (UPS). It was previously shown that rod photoreceptors in multiple mouse models of retinal degeneration suffer from proteostatic stress consisting of an insufficient cellular capacity for degrading UPS substrates. In this study, we focused on a specific UPS component required for the degradation of a subset of proteasome targets: the substrate-processing complex formed by the AAA+ ATPase P97/VCP and associated cofactors. To assess whether P97 capacity may be insufficient in degenerating rods, we employed two complementary in vivo proteasomal activity reporters whose degradation is either P97-dependent or P97-independent. Retinal accumulation of each reporter was measured in two models of retinal degeneration: the transducin γ -subunit knock-out ( Gγ 1 -/- ) and P23H rhodopsin knock-in (P23H) mice. Strikingly, the patterns of reporter accumulation differed between these models, indicating that the proteostatic stress observed in Gγ 1 -/- and P23H rods likely originates from different pathobiological mechanisms, in which UPS substrate degradation may or may not be limited by P97-dependent substrate processing. Further, we assessed whether P97 overexpression could ameliorate pathology in Gγ 1 -/- mice, in which proteostatic stress appears to result from P97 insufficiency. However, despite P97 overexpression being aphenotypic in other tissues, the ∼2.4-fold increase in retinal P97 content was toxic to rods, which complicated the interpretation of the observed phenotype. Our results highlight the complexity of pathophysiological mechanisms related to degrading misfolded proteins in mutant photoreceptors.
For decades, photoreceptors have been an outstanding model system for elucidating basic principles in sensory transduction and biochemistry and for understanding many facets of neuronal cell biology. In recent years, new knowledge of the kinetics of signaling and the large-scale movements of proteins underlying signaling has led to a deeper appreciation of the photoreceptor's unique challenge in mediating the first steps in vision over a wide range of light intensities. For decades, photoreceptors have been an outstanding model system for elucidating basic principles in sensory transduction and biochemistry and for understanding many facets of neuronal cell biology. In recent years, new knowledge of the kinetics of signaling and the large-scale movements of proteins underlying signaling has led to a deeper appreciation of the photoreceptor's unique challenge in mediating the first steps in vision over a wide range of light intensities. Retinal photoreceptors transduce information obtained in the form of absorbed photons into an electrical response that can be relayed across synapses to other neurons in the retina. In vertebrate photoreceptors, photon absorption and visual signaling take place in the outer segment (Fig. 1), a sensory cilium tightly packed with stacks of membranous discs containing extremely high densities of visual pigments and other signaling proteins. This morphological arrangement allows photons to be efficiently absorbed as they pass through the outer segment. The signal from activated visual pigment (rhodopsin in rods or cone opsins in cones) must then be sufficiently amplified to generate an electrical response that overcomes intrinsic noise. Transduction from the light-absorbing visual pigment into an electrical response utilizes a G protein signaling pathway termed the phototransduction cascade, which leads to a decrease in the second messenger cGMP and the closure of cGMP-sensitive cation channels. The resulting hyperpolarization transiently decreases the release of glutamate from the photoreceptor synaptic terminals, signaling the number of absorbed photons to the rest of the visual system. Remarkably, photoreceptors both detect low light levels (single photons in the case of rods) and continue to rapidly and reliably signal changes in light intensity as illuminance increases over 10 orders of magnitude during the course of a typical day. Phototransduction has been the subject of many comprehensive reviews (1Burns M.E. Baylor D.A. Annu. Rev. Neurosci. 2001; 24: 779-805Crossref PubMed Scopus (334) Google Scholar, 2Fain G.L. Matthews H.R. Cornwall M.C. Koutalos Y. Physiol. Rev. 2001; 81: 117-151Crossref PubMed Scopus (446) Google Scholar, 3Arshavsky V.Y. Lamb T.D. Pugh Jr., E.N. Annu. Rev. Physiol. 2002; 64: 153-187Crossref PubMed Scopus (506) Google Scholar, 4Fu Y. Yau K.W. Pflugers Arch. 2007; 454: 805-819Crossref PubMed Scopus (208) Google Scholar). Here, we provide a framework introduction and briefly summarize the latest findings that are shaping our understanding of this first step in vision. Phototransduction begins when a photon causes cis-trans-isomerization of the chromophore 11-cis-retinal, which induces a rapid conformational change to the protein's fully active form, R*. R* activates molecules of the G protein transducin by catalyzing GDP/GTP exchange on the transducin α-subunit, Gαt (Fig. 1). Gαt·GTP separates from the transducin βγ-subunits and binds to the γ-subunit of its effector, cGMP phosphodiesterase (PDE), 3The abbreviations used are: PDEphosphodiesteraseGCguanylate cyclaseGCAPGC-activating protein. which releases this subunit's inhibitory constraint on the catalytic α- and β-subunits of PDE. Activated PDE rapidly hydrolyzes cGMP, thereby reducing its concentration in the cytoplasm and causing cGMP-sensitive cation channels in the plasma membrane to close. The closure of channels reduces the inward cation current, resulting in a transient photoresponse generated within milliseconds (Fig. 2). phosphodiesterase guanylate cyclase GC-activating protein. The photoresponse persists until each phototransduction protein becomes deactivated through the action of one or more regulatory enzymes (Fig. 1). Specifically, each R* in rods must be phosphorylated at multiple C-terminal sites by rhodopsin kinase, with each added phosphate partially reducing the rate with which R* can activate transducin (5Maeda T. Imanishi Y. Palczewski K. Prog. Retin. Eye Res. 2003; 22: 417-434Crossref PubMed Scopus (125) Google Scholar). After the addition of three phosphates (6Vishnivetskiy S.A. Raman D. Wei J. Kennedy M.J. Hurley J.B. Gurevich V.V. J. Biol. Chem. 2007; 282: 32075-32083Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 7Doan T. Mendez A. Detwiler P.B. Chen J. Rieke F. Science. 2006; 313: 530-533Crossref PubMed Scopus (105) Google Scholar, 8Mendez A. Burns M.E. Roca A. Lem J. Wu L.W. Simon M.I. Baylor D.A. Chen J. Neuron. 2000; 28: 153-164Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar), arrestin (Arr1 in rods and Arr1 and Arr4 in cones) binds to R* with high affinity, completely blocking subsequent transducin activation. Likewise, transducin and PDE remain active until transducin hydrolyzes GTP. This hydrolysis is catalyzed by a triumvirate complex of proteins consisting of RGS9-1, Gβ5-L, and R9AP (the "RGS9 complex") (9Anderson G.R. Posokhova E. Martemyanov K.A. Cell Biochem. Biophys. 2009; 54: 33-46Crossref PubMed Scopus (109) Google Scholar). Finally, cGMP is restored through the action of guanylate cyclase (GC) (10Stephen R. Filipek S. Palczewski K. Sousa M.C. Photochem. Photobiol. 2008; 84: 903-910Crossref PubMed Scopus (41) Google Scholar). In normal mouse rods, the expression level of the RGS9 complex rate-limits the recovery of the rod's response to both single photons and bright flashes of light (11Krispel C.M. Chen D. Melling N. Chen Y.J. Martemyanov K.A. Quillinan N. Arshavsky V.Y. Wensel T.G. Chen C.K. Burns M.E. Neuron. 2006; 51: 409-416Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). Altering the expression level of R9AP changes the cellular content of the entire RGS9 complex (11Krispel C.M. Chen D. Melling N. Chen Y.J. Martemyanov K.A. Quillinan N. Arshavsky V.Y. Wensel T.G. Chen C.K. Burns M.E. Neuron. 2006; 51: 409-416Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 12Keresztes G. Martemyanov K.A. Krispel C.M. Mutai H. Yoo P.J. Maison S.F. Burns M.E. Arshavsky V.Y. Heller S. J. Biol. Chem. 2004; 279: 1581-1584Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Underexpression of R9AP reduces the level of the RGS9 complex and makes rod responses much slower to recover (11Krispel C.M. Chen D. Melling N. Chen Y.J. Martemyanov K.A. Quillinan N. Arshavsky V.Y. Wensel T.G. Chen C.K. Burns M.E. Neuron. 2006; 51: 409-416Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 13Burns M.E. Pugh Jr., E.N. Biophys. J. 2009; 97: 1538-1547Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar), whereas R9AP overexpression increases the RGS9 content and makes photoresponses recover much faster (11Krispel C.M. Chen D. Melling N. Chen Y.J. Martemyanov K.A. Quillinan N. Arshavsky V.Y. Wensel T.G. Chen C.K. Burns M.E. Neuron. 2006; 51: 409-416Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 14Gross O.P. Burns M.E. J. Neurosci. 2010; 30: 3450-3457Crossref PubMed Scopus (66) Google Scholar, 15Chen C.K. Woodruff M.L. Chen F.S. Chen D. Fain G.L. J. Neurosci. 2010; 30: 1213-1220Crossref PubMed Scopus (54) Google Scholar). Interestingly, the rod responses of every mammalian species examined so far recover with approximately the same time constant of ∼200 ms. This similarity suggests that the expression level of the RGS9 complex is tightly regulated and that there is some evolutionary pressure that sets this relatively slow time constant for rod vision. Notably, cones express more RGS9 than rods (16Cowan C.W. Fariss R.N. Sokal I. Palczewski K. Wensel T.G. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 5351-5356Crossref PubMed Scopus (147) Google Scholar, 17Zhang X. Wensel T.G. Kraft T.W. J. Neurosci. 2003; 23: 1287-1297Crossref PubMed Google Scholar), which is likely to contribute to their faster recovery. Indeed, recent physiology experiments with salamander cones suggest that the rate-limiting step for the recovery of cone responses is the deactivation of cone opsin (18Matthews H.R. Sampath A.P. J. Gen. Physiol. 2010; 135: 355-366Crossref PubMed Scopus (24) Google Scholar). The ability of rods to generate sizable responses to the absorption of single photons arises in part from the signal amplification conferred by the activation of many transducin molecules by a single R*. The rate of transducin activation in rods is ∼150 s−1 in cold-blooded vertebrates (19Leskov I.B. Klenchin V.A. Handy J.W. Whitlock G.G. Govardovskii V.I. Bownds M.D. Lamb T.D. Pugh Jr., E.N. Arshavsky V.Y. Neuron. 2000; 27: 525-537Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar) and 2–3-fold faster in mammals (20Heck M. Hofmann K.P. J. Biol. Chem. 2001; 276: 10000-10009Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). This rate is far higher than those measured in other G protein signaling pathways, undoubtedly because of the unusually high density of transducin on the disc membrane (∼1:8 molar ratio with rhodopsin in mice). However, despite the high rate of transducin activation, the number of activated Gαt molecules produced during the single photon response is actually surprisingly small, ∼10–15 in the mouse. This is because the effective lifetime of R* is relatively brief, ∼40 ms (13Burns M.E. Pugh Jr., E.N. Biophys. J. 2009; 97: 1538-1547Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 14Gross O.P. Burns M.E. J. Neurosci. 2010; 30: 3450-3457Crossref PubMed Scopus (66) Google Scholar), and even during the brief time before arrestin binds, the rate of transducin activation is being gradually diminished by sequential phosphorylations. In mouse rods, the brief effective lifetime of R* is set by the rate of its phosphorylation and by the concentration of arrestin (14Gross O.P. Burns M.E. J. Neurosci. 2010; 30: 3450-3457Crossref PubMed Scopus (66) Google Scholar). Remarkably, the concentration of arrestin available for R* binding is continuously buffered by its self-association into dimers and tetramers, neither of which can bind R* (21Hanson S.M. Van Eps N. Francis D.J. Altenbach C. Vishnivetskiy S.A. Arshavsky V.Y. Klug C.S. Hubbell W.L. Gurevich V.V. EMBO J. 2007; 26: 1726-1736Crossref PubMed Scopus (95) Google Scholar, 22Kim M. Hanson S.M. Vishnivetskiy S.A. Song X. Cleghorn W.M. Hubbell W.L. Gurevich V.V. Biochemistry. 2011; 50: 2235-2242Crossref PubMed Scopus (39) Google Scholar). Arrestin oligomerization not only regulates R* lifetime but helps to minimize the adverse consequences of high arrestin levels on photoreceptor viability (23Song X. Vishnivetskiy S.A. Seo J. Chen J. Gurevich E.V. Gurevich V.V. Neuroscience. 2011; 174: 37-49Crossref PubMed Scopus (62) Google Scholar). Rods and cones employ many mechanisms to avoid saturation by bright light and to adjust the amplitude and time course of their photoresponses to ever-changing ambient illumination, a process collectively known as light adaptation (Fig. 2) (2Fain G.L. Matthews H.R. Cornwall M.C. Koutalos Y. Physiol. Rev. 2001; 81: 117-151Crossref PubMed Scopus (446) Google Scholar, 24Pugh Jr., E.N. Nikonov S. Lamb T.D. Curr. Opin. Neurobiol. 1999; 9: 410-418Crossref PubMed Scopus (273) Google Scholar). Historically, most photoreceptor adaptation was thought to be mediated by the decline in intracellular Ca2+ that accompanies the photoresponse. The levels of Ca2+ fall in light because its influx is reduced when cGMP-gated channels close, whereas Ca2+ efflux via the Ca2+/K+/Na+ exchanger continues. The reduction in intracellular Ca2+ is sensed by several different Ca2+-binding proteins, including GC-activating proteins (GCAPs), which stimulate cGMP synthesis by GC when Ca2+ falls (10Stephen R. Filipek S. Palczewski K. Sousa M.C. Photochem. Photobiol. 2008; 84: 903-910Crossref PubMed Scopus (41) Google Scholar). The Ca2+/GCAP-dependent regulation of GC activity forms a powerful feedback mechanism in which the rate of cGMP synthesis increases as Ca2+ falls during the response to light, helping to restore cGMP levels rapidly and allowing the cGMP channels to reopen. There are two GC isoforms in photoreceptors, RetGC1 and RetGC2, and also several different GCAPs, with all vertebrates expressing at least two, GCAP1 and GCAP2; the relative expression levels of GC1/GC2 and GCAP1/GCAP2 are not equivalent in rods and cones (10Stephen R. Filipek S. Palczewski K. Sousa M.C. Photochem. Photobiol. 2008; 84: 903-910Crossref PubMed Scopus (41) Google Scholar). Likewise, the Ca2+ dependence of GC1 and GC2 regulation by GCAP1 and GCAP2 is different. However, the maximal ranges of each GC activity regulation by each GCAP are comparable (see discussion in Ref. 25Peshenko I.V. Olshevskaya E.V. Savchenko A.B. Karan S. Palczewski K. Baehr W. Dizhoor A.M. Biochemistry. 2011; 50: 5590-5600Crossref PubMed Scopus (73) Google Scholar). 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In cones, however, the effect is more rapid and has greater magnitude (46Rebrik T.I. Korenbrot J.I. J. Gen. Physiol. 1998; 112: 537-548Crossref PubMed Scopus (69) Google Scholar, 47Rebrik T.I. Kotelnikova E.A. Korenbrot J.I. J. Gen. Physiol. 2000; 116: 521-534Crossref PubMed Scopus (28) Google Scholar). Taken together, these Ca2+ feedback mechanisms account for most adaptation that occurs at low-to-moderate levels of light intensity, developing over a period of seconds. Ca2+-independent mechanisms also contribute to adaptation in photoreceptors, most notably the increased cGMP turnover in constant illumination (48Nikonov S. Lamb T.D. Pugh Jr., E.N. J. Gen. Physiol. 2000; 116: 795-824Crossref PubMed Scopus (126) Google Scholar). When the PDE activity is high in steady light, the activation of the same amount of PDE by a photon results in a smaller fractional change in the overall PDE activity. This produces a smaller and briefer change in cGMP, resulting in a smaller response that recovers more quickly. These contributions of the steady PDE activity to the amplitude and time course of photoresponses may not be intuitive and so have been reviewed using various physical analogies (48Nikonov S. Lamb T.D. Pugh Jr., E.N. J. Gen. Physiol. 2000; 116: 795-824Crossref PubMed Scopus (126) Google Scholar, 49Govardovskii V.I. Calvert P.D. Arshavsky V.Y. J. Gen. Physiol. 2000; 116: 791-794Crossref PubMed Scopus (23) Google Scholar). Finally, light adaptation has also been documented to occur at longer time scales of tens of seconds in both lower and higher vertebrates (50Krispel C.M. Chen C.K. Simon M.I. Burns M.E. J. Gen. Physiol. 2003; 122: 703-712Crossref PubMed Scopus (46) Google Scholar, 51Calvert P.D. Govardovskii V.I. Arshavsky V.Y. Makino C.L. J. Gen. Physiol. 2002; 119: 129-145Crossref PubMed Scopus (48) Google Scholar), although the underlying mechanisms remain unknown. A different type of adaptation mechanism induced by sustained bright light involves massive translocation of several phototransduction proteins between the outer segment and the rest of the photoreceptor cell (52Artemyev N.O. Mol. Neurobiol. 2008; 37: 44-51Crossref PubMed Scopus (38) Google Scholar, 53Slepak V.Z. Hurley J.B. IUBMB Life. 2008; 60: 2-9Crossref PubMed Scopus (68) Google Scholar, 54Calvert P.D. Strissel K.J. Schiesser W.E. Pugh Jr., E.N. Arshavsky V.Y. Trends Cell Biol. 2006; 16: 560-568Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 55Gurevich V.V. Hanson S.M. Song X. Vishnivetskiy S.A. Gurevich E.V. Prog. Retin. Eye Res. 2011; 30: 405-430Crossref PubMed Scopus (93) Google Scholar). Illumination causes significant fractions of transducin and recoverin to exit rod outer segments, whereas arrestin translocates in the opposite direction. These processes take place over the course of several minutes. The adaptive nature of transducin translocation in rods was demonstrated by experiments that correlated the loss of transducin from outer segments with a nearly 10-fold reduction in signal amplification in the phototransduction cascade (56Sokolov M. Lyubarsky A.L. Strissel K.J. Savchenko A.B. Govardovskii V.I. Pugh Jr., E.N. Arshavsky V.Y. Neuron. 2002; 34: 95-106Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar). This effect is likely mediated by the reduction in the rate of transducin activation by R* because this rate is dependent on the transducin concentration (19Leskov I.B. Klenchin V.A. Handy J.W. Whitlock G.G. Govardovskii V.I. Bownds M.D. Lamb T.D. Pugh Jr., E.N. Arshavsky V.Y. Neuron. 2000; 27: 525-537Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 20Heck M. Hofmann K.P. J. Biol. Chem. 2001; 276: 10000-10009Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). Although transducin translocation takes place at light intensities saturating rod light responses (see below), this reduction in signal amplification may be adaptive after the bright light is dimmed or extinguished, e.g. as dusk approaches, and vision is gradually switching from being cone-dominant to rod-dominant. Although still awaiting experimental validation, the functional role of translocation of other proteins is thought to be adaptive as well. Outer segments contain only a small fraction of total cellular arrestin in the dark (23Song X. Vishnivetskiy S.A. Seo J. Chen J. Gurevich E.V. Gurevich V.V. Neuroscience. 2011; 174: 37-49Crossref PubMed Scopus (62) Google Scholar, 57Strissel K.J. Sokolov M. Trieu L.H. Arshavsky V.Y. J. Neurosci. 2006; 26: 1146-1153Crossref PubMed Scopus (126) Google Scholar). As a result, phosphorylated R* produced by fairly low light levels could rapidly deplete the outer segment of free arrestin, slowing subsequent R* quenching. Thus, arrestin translocation provides a means to supply additional protein as needs arise upon illumination. Recoverin translocation from outer segments may also play an adaptive role by increasing the amount of rhodopsin kinase available to phosphorylate R*. This could contribute to light adaptation by speeding R* deactivation and reducing photoresponse sensitivity in a deeply light-adapted rod. A complementary role of protein translocation may be to protect rods from adverse effects of persistent light exposure (58Burns M.E. Arshavsky V.Y. Neuron. 2005; 48: 387-401Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 59Fain G.L. BioEssays. 2006; 28: 344-354Crossref PubMed Scopus (81) Google Scholar). In bright light, rods contribute little to vision, and instead of transducin running fruitlessly through the activation/deactivation cycle, it is stored away in a different cellular compartment to reduce energy consumption (60Chertov A.O. Holzhausen L. Kuok I.T. Couron D. 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Although diffusion may underlie the movement of proteins, the light-dependent changes in their distribution patterns are explained by the appearance or disappearance of specific protein-binding sites in individual subcellular compartments. For example,
Abstract The first steps in vision take place in photoreceptor cells, which are highly compartmentalized neurons exhibiting significant structural variation across species. The light-sensitive ciliary compartment, called the outer segment, is located atop of the cell soma, called the inner segment. In this study, we present an ultrastructural analysis of human photoreceptors, which reveals that, in contrast to this classic arrangement, the inner segment of human rods extends alongside the outer segment to form a structure hereby termed the “accessory inner segment”. While reminiscent of the actin-based microvilli known as “calyceal processes” observed in other species, the accessory inner segment is a unique structure: (1) it contains an extensive microtubule-based cytoskeleton, (2) it extends far alongside the outer segment, (3) its diameter is comparable to that of the outer segment, (4) it contains numerous mitochondria, and (5) it forms electron-dense structures that likely mediate adhesion to the outer segment. Given that the spacing of extrafoveal human photoreceptors is more sparse than in non-primate species, with vast amounts of interphotoreceptor matrix present between cells, the closely apposed accessory inner segment likely provides structural support to the outer segment. This discovery expands our understanding of the human retina and directs future studies of human photoreceptor function in health and disease.
The remarkable ability of our vision to function under ever-changing conditions of ambient illumination is mediated by multiple molecular mechanisms regulating the light sensitivity of rods and cones. One such mechanism involves massive translocation of signaling proteins, including the G-protein transducin, into and out of the light-sensitive photoreceptor outer segment compartment. Transducin translocation extends the operating range of rods, but in cones transducin never translocates, which is puzzling because cones typically function in much brighter light than rods. Using genetically manipulated mice in which the rates of transducin activation and inactivation were altered, we demonstrate that, like in rods, transducin translocation in cones can be triggered when transducin activation exceeds a critical level, essentially saturating the photoresponse. However, this level is never achieved in wild-type cones: their superior ability to tightly control the rates of transducin activation and inactivation, responsible for avoiding saturation by light, also accounts for the prevention of transducin translocation at any light intensity.
