Publications by authors named "Mark W Hankins"

59 Publications

The functional characteristics of optogenetic gene therapy for vision restoration.

Cell Mol Life Sci 2021 Feb 29;78(4):1597-1613. Epub 2020 Jul 29.

The Nuffield Laboratory of Ophthalmology, Sleep and Circadian Neuroscience Institute, Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, UK.

Optogenetic strategies to restore vision in patients blind from end-stage retinal degenerations aim to render remaining retinal neurons light-sensitive. We present an innovative combination of multi-electrode array recordings together with a complex pattern-generating light source as a toolset to determine the extent to which neural retinal responses to complex light stimuli can be restored following viral delivery of red-shifted channelrhodopsin in the retinally degenerated mouse. Our data indicate that retinal output level spatiotemporal response characteristics achieved by optogenetic gene therapy closely parallel those observed for normal mice but equally reveal important limitations, some of which could be mitigated using bipolar-cell targeted gene-delivery approaches. As clinical trials are commencing, these data provide important new information on the capacity and limitations of channelrhodopsin-based gene therapies. The toolset we established enables comparing optogenetic constructs and stem-cell-based techniques, thereby providing an efficient and sensitive starting point to identify future approaches for vision restoration.
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http://dx.doi.org/10.1007/s00018-020-03597-6DOI Listing
February 2021

Expression and Localization of Kcne2 in the Vertebrate Retina.

Invest Ophthalmol Vis Sci 2020 03;61(3):33

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Purpose: To characterize the retinal expression and localization of Kcne2, an ancillary (β) ion-channel subunit with an important role in fine-tuning cellular excitability.

Methods: We analyzed available single-cell transcriptome data from tens of thousands of murine retinal cells for cell-type-specific expression of Kcne2 using state-of-the-art bioinformatics techniques. This evidence at the transcriptome level was complemented with a comprehensive immunohistochemical characterization of mouse retina (C57BL/6, ages 8-12 weeks) employing co-labeling techniques and cell-type-specific antibody markers. We furthermore examined how conserved the Kcne2 localization pattern in the retina was across species by performing immunostaining on zebrafish, cowbird, sheep, mice, and macaque.

Results: Kcne2 is distinctly expressed in cone photoreceptors and rod bipolar cells. At a subcellular level, the bulk of Kcne2 immunoreactivity can be observed in the outer plexiform layer. Here, it localizes into cone pedicles and likely the postsynaptic membrane of the rod bipolar cells. Thus, the vast majority of Kcne2 immunoreactivity is observed in a thin band in the outer plexiform layer. In addition to this, faint Kcne2 immunoreactivity can also be observed in cone inner segments and the somata of a small subset of cone ON bipolar cells. Strikingly, the localization of Kcne2 in the outer plexiform layer was preserved among all of the species studied, spanning at least 300 million years of evolution of the vertebrate kingdom.

Conclusions: The data we present here suggest an important and specific role for Kcne2 in the highly specialized photoreceptor-bipolar cell synapse.
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http://dx.doi.org/10.1167/iovs.61.3.33DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7401445PMC
March 2020

Defining the impact of melanopsin missense polymorphisms using in vivo functional rescue.

Hum Mol Genet 2018 08;27(15):2589-2603

Nuffield Department of Clinical Neurosciences, Sleep and Circadian Neuroscience Institute, OMPI G, Sir William Dunn School of Pathology, University of Oxford, Oxford, UK.

Melanopsin (OPN4) is an opsin photopigment expressed within intrinsically photosensitive retinal ganglion cells (ipRGCs) that mediate non-image forming (NIF) responses to light. Two single-nucleotide polymorphisms (SNPs) in human melanopsin (hOPN4), Pro10Leu and Thr394Ile, have recently been associated with abnormal NIF responses to light, including seasonal affective disorder. It has been suggested these behavioural changes are due to altered melanopsin signalling. However, there is currently no direct evidence to support this. Here we have used ipRGC-specific delivery of hOPN4 wild-type (WT), Pro10Leu or Thr394Ile adeno-associated viruses (AAV) to determine the functional consequences of hOPN4 SNPs on melanopsin-driven light responses and associated behaviours. Immunohistochemistry confirmed hOPN4 AAVs exclusively transduced mouse ipRGCs. Behavioural phenotyping performed before and after AAV injection demonstrated that both hOPN4 Pro10Leu and Thr394Ile could functionally rescue pupillary light responses and circadian photoentrainment in Opn4-/- mice, with no differences in NIF behaviours detected for animals expressing either SNP compared to hOPN4 WT. Multi-electrode array recordings revealed that ipRGCs expressing hOPN4 Thr394Ile exhibit melanopsin-driven light responses with significantly attenuated response amplitude, decreased sensitivity and faster offset kinetics compared to hOPN4 WT. IpRGCs expressing hOpn4 Pro10Leu also showed reduced response amplitude. Collectively these data suggest Thr394Ile and Pro10Leu may be functionally significant SNPs, which result in altered melanopsin signalling. To our knowledge, this study provides the first direct evidence for the effects of hOPN4 polymorphisms on melanopsin-driven light responses and NIF behaviours in vivo, providing further insight into the role of these SNPs in melanopsin function and human physiology.
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http://dx.doi.org/10.1093/hmg/ddy150DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6048994PMC
August 2018

Functional characterisation of naturally occurring mutations in human melanopsin.

Cell Mol Life Sci 2018 Oct 26;75(19):3609-3624. Epub 2018 Apr 26.

Nuffield Laboratory of Ophthalmology, Sleep and Circadian Neuroscience Institute, Nuffield Department of Clinical Neurosciences, Sir William Dunn School of Pathology, University of Oxford, OMPI G, South Parks Road, Oxford, OX1 3RE, UK.

Melanopsin is a blue light-sensitive opsin photopigment involved in a range of non-image forming behaviours, including circadian photoentrainment and the pupil light response. Many naturally occurring genetic variants exist within the human melanopsin gene (OPN4), yet it remains unclear how these variants affect melanopsin protein function and downstream physiological responses to light. Here, we have used bioinformatic analysis and in vitro expression systems to determine the functional phenotypes of missense human OPN4 variants. From 1242 human OPN4 variants collated in the NCBI Short Genetic Variation database (dbSNP), we identified 96 that lead to non-synonymous amino acid substitutions. These 96 missense mutations were screened using sequence alignment and comparative approaches to select 16 potentially deleterious variants for functional characterisation using calcium imaging of melanopsin-driven light responses in HEK293T cells. We identify several previously uncharacterised OPN4 mutations with altered functional properties, including attenuated or abolished light responses, as well as variants demonstrating abnormal response kinetics. These data provide valuable insight into the structure-function relationships of human melanopsin, including several key functional residues of the melanopsin protein. The identification of melanopsin variants with significantly altered function may serve to detect individuals with disrupted melanopsin-based light perception, and potentially highlight those at increased risk of sleep disturbance, circadian dysfunction, and visual abnormalities.
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http://dx.doi.org/10.1007/s00018-018-2813-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6133154PMC
October 2018

Differential roles for cryptochromes in the mammalian retinal clock.

FASEB J 2018 08 21;32(8):4302-4314. Epub 2018 Mar 21.

Sleep and Circadian Neuroscience Institute, University of Oxford, Oxford, United Kingdom.

Cryptochromes 1 and 2 (CRY1/2) are key components of the negative limb of the mammalian circadian clock. Like many peripheral tissues, Cry1 and -2 are expressed in the retina, where they are thought to play a role in regulating rhythmic physiology. However, studies differ in consensus as to their localization and function, and CRY1 immunostaining has not been convincingly demonstrated in the retina. Here we describe the expression and function of CRY1 and -2 in the mouse retina in both sexes. Unexpectedly, we show that CRY1 is expressed throughout all retinal layers, whereas CRY2 is restricted to the photoreceptor layer. Retinal period 2::luciferase recordings from CRY1-deficient mice show reduced clock robustness and stability, while those from CRY2-deficient mice show normal, albeit long-period, rhythms. In functional studies, we then investigated well-defined rhythms in retinal physiology. Rhythms in the photopic electroretinogram, contrast sensitivity, and pupillary light response were all severely attenuated or abolished in CRY1-deficient mice. In contrast, these physiological rhythms are largely unaffected in mice lacking CRY2, and only photopic electroretinogram rhythms are affected. Together, our data suggest that CRY1 is an essential component of the mammalian retinal clock, whereas CRY2 has a more limited role.-Wong, J. C. Y., Smyllie, N. J., Banks, G. T., Pothecary, C. A., Barnard, A. R., Maywood, E. S., Jagannath, A., Hughes, S., van der Horst, G. T. J., MacLaren, R. E., Hankins, M. W., Hastings, M. H., Nolan, P. M., Foster, R. G., Peirson, S. N. Differential roles for cryptochromes in the mammalian retinal clock.
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http://dx.doi.org/10.1096/fj.201701165RRDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6071063PMC
August 2018

Long-term restoration of visual function in end-stage retinal degeneration using subretinal human melanopsin gene therapy.

Proc Natl Acad Sci U S A 2017 10 2;114(42):11211-11216. Epub 2017 Oct 2.

Nuffield Laboratory of Ophthalmology, Nuffield Department of Clinical Neurosciences, University of Oxford, National Institute for Health Research Biomedical Research Centre, Oxford OX3 9DU, United Kingdom;

Optogenetic strategies to restore vision in patients who are blind from end-stage retinal degenerations aim to render remaining retinal cells light sensitive once photoreceptors are lost. Here, we assessed long-term functional outcomes following subretinal delivery of the human melanopsin gene (OPN4) in the mouse model of retinal degeneration using an adeno-associated viral vector. Ectopic expression of OPN4 using a ubiquitous promoter resulted in cellular depolarization and ganglion cell action potential firing. Restoration of the pupil light reflex, behavioral light avoidance, and the ability to perform a task requiring basic image recognition were restored up to 13 mo following injection. These data suggest that melanopsin gene therapy via a subretinal route may be a viable and stable therapeutic option for the treatment of end-stage retinal degeneration in humans.
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http://dx.doi.org/10.1073/pnas.1701589114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5651734PMC
October 2017

Expression and localisation of two-pore domain (K2P) background leak potassium ion channels in the mouse retina.

Sci Rep 2017 04 26;7:46085. Epub 2017 Apr 26.

The Nuffield Laboratory of Ophthalmology, Sleep and Circadian Neuroscience Institute, Nuffield Department of Clinical Neurosciences, University of Oxford, Sir William Dunn School of Pathology, OMPI G, South Parks Road, Oxford, OX1 3RE, UK.

Two-pore domain (K2P) potassium channels perform essential roles in neuronal function. These channels produce background leak type potassium currents that act to regulate resting membrane potential and levels of cellular excitability. 15 different K2P channels have been identified in mammals and these channels perform important roles in a wide number of physiological systems. However, to date there is only limited data available concerning the expression and role of K2P channels in the retina. In this study we conduct the first comprehensive study of K2P channel expression in the retina. Our data show that K2P channels are widely expressed in the mouse retina, with variations in expression detected at different times of day and throughout postnatal development. The highest levels of K2P channel expression are observed for Müller cells (TWIK-1, TASK-3, TRAAK, and TREK-2) and retinal ganglion cells (TASK-1, TREK-1, TWIK-1, TWIK-2 and TWIK-3). These data offer new insight into the channels that regulate the resting membrane potential and electrical activity of retinal cells, and suggests that K2P channels are well placed to act as central regulators of visual signalling pathways. The prominent role of K2P channels in neuroprotection offers novel avenues of research into the treatment of common retinal diseases.
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http://dx.doi.org/10.1038/srep46085DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5405414PMC
April 2017

Constant Light Desynchronizes Olfactory versus Object and Visuospatial Recognition Memory Performance.

J Neurosci 2017 03 6;37(13):3555-3567. Epub 2017 Mar 6.

Sleep and Circadian Neuroscience Institute (SCNi), Nuffield Department of Clinical Neurosciences, Oxford Molecular Pathology Institute, Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, United Kingdom,

Circadian rhythms optimize physiology and behavior to the varying demands of the 24 h day. The master circadian clock is located in the suprachiasmatic nuclei (SCN) of the hypothalamus and it regulates circadian oscillators in tissues throughout the body to prevent internal desynchrony. Here, we demonstrate for the first time that, under standard 12 h:12 h light/dark (LD) cycles, object, visuospatial, and olfactory recognition performance in C57BL/6J mice is consistently better at midday relative to midnight. However, under repeated exposure to constant light (LL), recognition performance becomes desynchronized, with object and visuospatial performance better at subjective midday and olfactory performance better at subjective midnight. This desynchrony in behavioral performance is mirrored by changes in expression of the canonical clock genes and ( and ), as well as the immediate-early gene in the SCN, dorsal hippocampus, and olfactory bulb. Under LL, rhythmic and expression is attenuated in the SCN. In contrast, hippocampal gene expression remains rhythmic, mirroring object and visuospatial performance. Strikingly, and expression in the olfactory bulb is reversed, mirroring the inverted olfactory performance. Temporal desynchrony among these regions does not result in arrhythmicity because core body temperature and exploratory activity rhythms persist under LL. Our data provide the first demonstration that abnormal lighting conditions can give rise to temporal desynchrony between autonomous circadian oscillators in different regions, with different consequences for performance across different sensory domains. Such a dispersed network of dissociable circadian oscillators may provide greater flexibility when faced with conflicting environmental signals. A master circadian clock in the suprachiasmatic nuclei (SCN) of the hypothalamus regulates physiology and behavior across the 24 h day by synchronizing peripheral clocks throughout the brain and body. Without the SCN, these peripheral clocks rapidly become desynchronized. Here, we provide a unique demonstration that, under lighting conditions in which the central clock in the SCN is dampened, peripheral oscillators in the hippocampus and olfactory bulb become desynchronized, along with the behavioral processes mediated by these clocks. Multiple clocks that adopt different phase relationships may enable processes occurring in different brain regions to be optimized to specific phases of the 24 h day. Moreover, such a dispersed network of dissociable circadian clocks may provide greater flexibility when faced with conflicting environmental signals (e.g., seasonal changes in photoperiod).
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http://dx.doi.org/10.1523/JNEUROSCI.3213-16.2017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5373134PMC
March 2017

Modulation of recognition memory performance by light requires both melanopsin and classical photoreceptors.

Proc Biol Sci 2016 12;283(1845)

Sleep and Circadian Neuroscience Institute (Nuffield Department of Clinical Neurosciences), Oxford Molecular Pathology Institute, Sir William Dunn School of Pathology, South Parks Road, Oxford OX1 3RE, UK

Acute light exposure exerts various effects on physiology and behaviour. Although the effects of light on brain network activity in humans are well demonstrated, the effects of light on cognitive performance are inconclusive, with the size, as well as direction, of the effect depending on the nature of the task. Similarly, in nocturnal rodents, bright light can either facilitate or disrupt performance depending on the type of task employed. Crucially, it is unclear whether the effects of light on behavioural performance are mediated via the classical image-forming rods and cones or the melanopsin-expressing photosensitive retinal ganglion cells. Here, we investigate the modulatory effects of light on memory performance in mice using the spontaneous object recognition task. Importantly, we examine which photoreceptors are required to mediate the effects of light on memory performance. By using a cross-over design, we show that object recognition memory is disrupted when the test phase is conducted under a bright light (350 lux), regardless of the light level in the sample phase (10 or 350 lux), demonstrating that exposure to a bright light at the time of test, rather than at the time of encoding, impairs performance. Strikingly, the modulatory effect of light on memory performance is completely abolished in both melanopsin-deficient and rodless-coneless mice. Our findings provide direct evidence that melanopsin-driven and rod/cone-driven photoresponses are integrated in order to mediate the effect of light on memory performance.
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http://dx.doi.org/10.1098/rspb.2016.2275DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5204172PMC
December 2016

Single residue AAV capsid mutation improves transduction of photoreceptors in the Abca4 mouse and bipolar cells in the rd1 mouse and human retina ex vivo.

Gene Ther 2016 11 14;23(11):767-774. Epub 2016 Jul 14.

Nuffield Laboratory of Ophthalmology, University of Oxford, NIHR Biomedical Research Centre, UK.

Gene therapy using adeno-associated viral (AAV) vectors for the treatment of retinal degenerations has shown safety and efficacy in clinical trials. However, very high levels of vector expression may be necessary for the treatment of conditions such as Stargardt disease where a dual vector approach is potentially needed, or in optogenetic strategies for end-stage degeneration in order to achieve maximal light sensitivity. In this study, we assessed two vectors with single capsid mutations, rAAV2/2(Y444F) and rAAV2/8(Y733F) in their ability to transduce retina in the Abca4 and rd1 mouse models of retinal degeneration. We noted significantly increased photoreceptor transduction using rAAV2/8(Y733F) in the Abca4 mouse, in contrast to previous work where vectors tested in this model have shown low levels of photoreceptor transduction. Bipolar cell transduction was achieved following subretinal delivery of both vectors in the rd1 mouse, and via intravitreal delivery of rAAV2/2(Y444F). The successful use of rAAV2/8(Y733F) to target bipolar cells was further validated on human tissue using an ex vivo culture system of retinal explants. Capsid mutant AAV vectors transduce human retinal cells and may be particularly suited to treat retinal degenerations in which high levels of transgene expression are required.
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http://dx.doi.org/10.1038/gt.2016.54DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5097463PMC
November 2016

Characterisation of light responses in the retina of mice lacking principle components of rod, cone and melanopsin phototransduction signalling pathways.

Sci Rep 2016 06 15;6:28086. Epub 2016 Jun 15.

The Nuffield Laboratory of Ophthalmology, Sleep and Circadian Neuroscience Institute, Nuffield Department of Clinical Neurosciences, University of Oxford, Sir William Dunn School of Pathology, OMPI G, South Parks Road, Oxford, OX1 3RE, UK.

Gnat(-/-), Cnga3(-/-), Opn4(-/-) triple knockout (TKO) mice lack essential components of phototransduction signalling pathways present in rods, cones and photosensitive retinal ganglion cells (pRGCs), and are therefore expected to lack all sensitivity to light. However, a number of studies have shown that light responses persist in these mice. In this study we use multielectrode array (MEA) recordings and light-induced c-fos expression to further characterise the light responses of the TKO retina. Small, but robust electroretinogram type responses are routinely detected during MEA recordings, with properties consistent with rod driven responses. Furthermore, a distinctive pattern of light-induced c-fos expression is evident in the TKO retina, with c-fos expression largely restricted to a small subset of amacrine cells that express disabled-1 (Dab1) but lack expression of glycine transporter-1 (GlyT-1). Collectively these data are consistent with the persistence of a novel light sensing pathway in the TKO retina that originates in rod photoreceptors, potentially a rare subset of rods with distinct functional properties, and which is propagated to an atypical subtype of AII amacrine cells. Furthermore, the minimal responses observed following UV light stimulation suggest only a limited role for the non-visual opsin OPN5 in driving excitatory light responses within the mouse retina.
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http://dx.doi.org/10.1038/srep28086DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4908426PMC
June 2016

Melanopsin Regulates Both Sleep-Promoting and Arousal-Promoting Responses to Light.

PLoS Biol 2016 06 8;14(6):e1002482. Epub 2016 Jun 8.

Sleep and Circadian Neuroscience Institute (SCNi), Nuffield Department of Clinical Neurosciences, Oxford Molecular Pathology Institute, Dunn School of Pathology, University of Oxford, Oxford, United Kingdom.

Light plays a critical role in the regulation of numerous aspects of physiology and behaviour, including the entrainment of circadian rhythms and the regulation of sleep. These responses involve melanopsin (OPN4)-expressing photosensitive retinal ganglion cells (pRGCs) in addition to rods and cones. Nocturnal light exposure in rodents has been shown to result in rapid sleep induction, in which melanopsin plays a key role. However, studies have also shown that light exposure can result in elevated corticosterone, a response that is not compatible with sleep. To investigate these contradictory findings and to dissect the relative contribution of pRGCs and rods/cones, we assessed the effects of light of different wavelengths on behaviourally defined sleep. Here, we show that blue light (470 nm) causes behavioural arousal, elevating corticosterone and delaying sleep onset. By contrast, green light (530 nm) produces rapid sleep induction. Compared to wildtype mice, these responses are altered in melanopsin-deficient mice (Opn4-/-), resulting in enhanced sleep in response to blue light but delayed sleep induction in response to green or white light. We go on to show that blue light evokes higher Fos induction in the SCN compared to the sleep-promoting ventrolateral preoptic area (VLPO), whereas green light produced greater responses in the VLPO. Collectively, our data demonstrates that nocturnal light exposure can have either an arousal- or sleep-promoting effect, and that these responses are melanopsin-mediated via different neural pathways with different spectral sensitivities. These findings raise important questions relating to how artificial light may alter behaviour in both the work and domestic setting.
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http://dx.doi.org/10.1371/journal.pbio.1002482DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4898879PMC
June 2016

An extended family of novel vertebrate photopigments is widely expressed and displays a diversity of function.

Genome Res 2015 Nov 8;25(11):1666-79. Epub 2015 Oct 8.

Nuffield Laboratory of Ophthalmology, Nuffield Department of Clinical Neurosciences, John Radcliffe Hospital, University of Oxford, Oxford, OX3 9DU, United Kingdom;

Light affects animal physiology and behavior more than simply through classical visual, image-forming pathways. Nonvisual photoreception regulates numerous biological systems, including circadian entrainment, DNA repair, metabolism, and behavior. However, for the majority of these processes, the photoreceptive molecules involved are unknown. Given the diversity of photophysiological responses, the question arises whether a single photopigment or a greater diversity of proteins within the opsin superfamily detect photic stimuli. Here, a functional genomics approach identified the full complement of photopigments in a highly light-sensitive model vertebrate, the zebrafish (Danio rerio), and characterized their tissue distribution, expression levels, and biochemical properties. The results presented here reveal the presence of 42 distinct genes encoding 10 classical visual photopigments and 32 nonvisual opsins, including 10 novel opsin genes comprising four new pigment classes. Consistent with the presence of light-entrainable circadian oscillators in zebrafish, all adult tissues examined expressed two or more opsins, including several novel opsins. Spectral and electrophysiological analyses of the new opsins demonstrate that they form functional photopigments, each with unique chromophore-binding and wavelength specificities. This study has revealed a remarkable number and diversity of photopigments in zebrafish, the largest number so far discovered for any vertebrate. Found in amphibians, reptiles, birds, and all three mammalian clades, most of these genes are not restricted to teleosts. Therefore, nonvisual light detection is far more complex than initially appreciated, which has significant biological implications in understanding photoreception in vertebrates.
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http://dx.doi.org/10.1101/gr.189886.115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4617963PMC
November 2015

Isoforms of Melanopsin Mediate Different Behavioral Responses to Light.

Curr Biol 2015 Sep 27;25(18):2430-4. Epub 2015 Aug 27.

Nuffield Laboratory of Ophthalmology, John Radcliffe Hospital, University of Oxford, Levels 5-6 West Wing, Headley Way, Oxford OX3 9DU, UK. Electronic address:

Melanopsin (OPN4) is a retinal photopigment that mediates a wide range of non-image-forming (NIF) responses to light including circadian entrainment, sleep induction, the pupillary light response (PLR), and negative masking of locomotor behavior (the acute suppression of activity in response to light). How these diverse NIF responses can all be mediated by a single photopigment has remained a mystery. We reasoned that the alternative splicing of melanopsin could provide the basis for functionally distinct photopigments arising from a single gene. The murine melanopsin gene is indeed alternatively spliced, producing two distinct isoforms, a short (OPN4S) and a long (OPN4L) isoform, which differ only in their C terminus tails. Significantly, both isoforms form fully functional photopigments. Here, we show that different isoforms of OPN4 mediate different behavioral responses to light. By using RNAi-mediated silencing of each isoform in vivo, we demonstrated that the short isoform (OPN4S) mediates light-induced pupillary constriction, the long isoform (OPN4L) regulates negative masking, and both isoforms contribute to phase-shifting circadian rhythms of locomotor behavior and light-mediated sleep induction. These findings demonstrate that splice variants of a single receptor gene can regulate strikingly different behaviors.
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http://dx.doi.org/10.1016/j.cub.2015.07.071DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4580334PMC
September 2015

Photic regulation of clock systems.

Methods Enzymol 2015 26;552:125-43. Epub 2014 Dec 26.

Nuffield Laboratory of Ophthalmology, Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, United Kingdom. Electronic address:

Circadian rhythms in physiology and behavior provide a selective advantage by enabling organisms to anticipate rhythmic changes in their environment. These rhythms are based upon a molecular clock generated via an intracellular transcriptional-translational feedback loop involving a number of key clock genes. However, to be of practical use, circadian rhythms need to be entrained to the external environment. In mammals, the primary signal for entrainment is light detected by the photoreceptors of the eye. Research on the mechanisms of photic entrainment has identified a novel photoreceptor system in the retina, consisting of photosensitive retinal ganglion cells expressing the photopigment melanopsin. Light input from these retinal photoreceptors reaches the master circadian pacemaker in the suprachiasmatic nuclei (SCN) via the retinohypothalamic tract, where it then interacts with the molecular clock to bring about entrainment. This chapter focuses on the retinal photoreceptors mediating entrainment, and how light information from the retina is transmitted to the SCN, before detailing recent advances in our understanding of how the molecular clock within the SCN is regulated by light input. Finally, the primary assays that have been used to measure photic entrainment are described.
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http://dx.doi.org/10.1016/bs.mie.2014.10.018DOI Listing
November 2015

Vision: melanopsin as a novel irradiance detector at the heart of vision.

Curr Biol 2014 Nov 3;24(21):R1055-7. Epub 2014 Nov 3.

Nuffield Laboratory of Ophthalmology, NDCN, University of Oxford, Oxford, OX3 9DU, UK.

A recent study defines a novel role of melanopsin-expressing ipRGCs, showing that these inner retinal photoreceptors function as retinal irradiance detectors and provide a local measure of luminance to regulate functional adaptation in the mammalian retina.
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http://dx.doi.org/10.1016/j.cub.2014.09.034DOI Listing
November 2014

The hypothalamic photoreceptors regulating seasonal reproduction in birds: a prime role for VA opsin.

Front Neuroendocrinol 2015 Apr 22;37:13-28. Epub 2014 Nov 22.

Sleep and Circadian Neuroscience Institute, Nuffield Laboratory of Ophthalmology, Nuffield Department of Clinical Neurosciences, University of Oxford, Headley Way, Oxford OX3 9DU, United Kingdom. Electronic address:

Extraretinal photoreceptors located within the medio-basal hypothalamus regulate the photoperiodic control of seasonal reproduction in birds. An action spectrum for this response describes an opsin photopigment with a λmax of ∼ 492 nm. Beyond this however, the specific identity of the photopigment remains unresolved. Several candidates have emerged including rod-opsin; melanopsin (OPN4); neuropsin (OPN5); and vertebrate ancient (VA) opsin. These contenders are evaluated against key criteria used routinely in photobiology to link orphan photopigments to specific biological responses. To date, only VA opsin can easily satisfy all criteria and we propose that this photopigment represents the prime candidate for encoding daylength and driving seasonal breeding in birds. We also show that VA opsin is co-expressed with both gonadotropin-releasing hormone (GnRH) and arginine-vasotocin (AVT) neurons. These new data suggest that GnRH and AVT neurosecretory pathways are endogenously photosensitive and that our current understanding of how these systems are regulated will require substantial revision.
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http://dx.doi.org/10.1016/j.yfrne.2014.11.001DOI Listing
April 2015

Using siRNA to define functional interactions between melanopsin and multiple G Protein partners.

Cell Mol Life Sci 2015 Jan 24;72(1):165-79. Epub 2014 Jun 24.

Nuffield Laboratory of Ophthalmology, University of Oxford, Oxford, OX3 9DU, UK.

Melanopsin expressing photosensitive retinal ganglion cells (pRGCs) represent a third class of ocular photoreceptors and mediate a range of non-image forming responses to light. Melanopsin is a G protein coupled receptor (GPCR) and existing data suggest that it employs a membrane bound signalling cascade involving Gnaq/11 type G proteins. However, to date the precise identity of the Gα subunits involved in melanopsin phototransduction remains poorly defined. Here we show that Gnaq, Gna11 and Gna14 are highly co-expressed in pRGCs of the mouse retina. Furthermore, using RNAi based gene silencing we show that melanopsin can signal via Gnaq, Gna11 or Gna14 in vitro, and demonstrate that multiple members of the Gnaq/11 subfamily, including Gna14 and at least Gnaq or Gna11, can participate in melanopsin phototransduction in vivo and contribute to the pupillary light responses of mice lacking rod and cone photoreceptors. This diversity of G protein interactions suggests additional complexity in the melanopsin phototransduction cascade and may provide a basis for generating the diversity of light responses observed from pRGC subtypes.
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http://dx.doi.org/10.1007/s00018-014-1664-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4282707PMC
January 2015

The CRTC1-SIK1 pathway regulates entrainment of the circadian clock.

Cell 2013 Aug;154(5):1100-1111

Nuffield Department of Clinical Neurosciences (Nuffield Laboratory of Ophthalmology), University of Oxford, Levels 5-6 West Wing, John Radcliffe Hospital, Headley Way, Oxford OX3 9DU, UK. Electronic address:

Retinal photoreceptors entrain the circadian system to the solar day. This photic resetting involves cAMP response element binding protein (CREB)-mediated upregulation of Per genes within individual cells of the suprachiasmatic nuclei (SCN). Our detailed understanding of this pathway is poor, and it remains unclear why entrainment to a new time zone takes several days. By analyzing the light-regulated transcriptome of the SCN, we have identified a key role for salt inducible kinase 1 (SIK1) and CREB-regulated transcription coactivator 1 (CRTC1) in clock re-setting. An entrainment stimulus causes CRTC1 to coactivate CREB, inducing the expression of Per1 and Sik1. SIK1 then inhibits further shifts of the clock by phosphorylation and deactivation of CRTC1. Knockdown of Sik1 within the SCN results in increased behavioral phase shifts and rapid re-entrainment following experimental jet lag. Thus SIK1 provides negative feedback, acting to suppress the effects of light on the clock. This pathway provides a potential target for the regulation of circadian rhythms.
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http://dx.doi.org/10.1016/j.cell.2013.08.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3898689PMC
August 2013

Nonuniform distribution and spectral tuning of photosensitive retinal ganglion cells of the mouse retina.

Curr Biol 2013 Sep 15;23(17):1696-701. Epub 2013 Aug 15.

Nuffield Laboratory of Ophthalmology, Nuffield Department of Clinical Neurosciences, University of Oxford, Headley Way, Oxford OX3 9DU, UK.

Melanopsin-expressing photosensitive retinal ganglion cells (pRGCs) represent a third class of retinal photoreceptor. These cells are intrinsically photosensitive, but also receive inputs from rod and cone photoreceptors, acting as the primary sensory conduit mediating non-image-forming responses to light. Multiple subtypes of pRGC have been described in the mouse retina with characteristic morphologies and functional properties, and which perform distinct physiological roles. Here, we examine the levels of melanopsin expression and distribution of pRGC subtypes across the mouse retina, identifying a previously unreported anatomical and functional specialization of the melanopsin system. Our results show a dorsal-ventral gradient in the expression of melanopsin and the distribution of pRGCs, which, combined with dorsal-ventral gradients in ultraviolet-sensitive and medium-wavelength-sensitive cone opsin expression, produce dramatic variations in the ratio of cone opsins and pRGCs across the retina. Using c-fos expression as a marker of light activation in vivo, we show that the responses of pRGCs are spectrally tuned by gradients in cone opsin expression depending on their location in the retina. These data illustrate the importance of classical photoreceptors in providing spectral tuning of pRGC light responses and have important implications for the complexity of non-image-forming responses to light.
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http://dx.doi.org/10.1016/j.cub.2013.07.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3770899PMC
September 2013

Assessment of tropism and effectiveness of new primate-derived hybrid recombinant AAV serotypes in the mouse and primate retina.

PLoS One 2013 9;8(4):e60361. Epub 2013 Apr 9.

Nuffield Laboratory of Ophthalmology, Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, United Kingdom.

Adeno-associated viral vectors (AAV) have been shown to be safe in the treatment of retinal degenerations in clinical trials. Thus, improving the efficiency of viral gene delivery has become increasingly important to increase the success of clinical trials. In this study, structural domains of different rAAV serotypes isolated from primate brain were combined to create novel hybrid recombinant AAV serotypes, rAAV2/rec2 and rAAV2/rec3. The efficacy of these novel serotypes were assessed in wild type mice and in two models of retinal degeneration (the Abca4(-/-) mouse which is a model for Stargardt disease and in the Pde6b(rd1/rd1) mouse) in vivo, in primate tissue ex-vivo, and in the human-derived SH-SY5Y cell line, using an identical AAV2 expression cassette. We show that these novel hybrid serotypes can transduce retinal tissue in mice and primates efficiently, although no more than AAV2/2 and rAAV2/5 serotypes. Transduction efficiency appeared lower in the Abca4(-/-) mouse compared to wild type with all vectors tested, suggesting an effect of specific retinal diseases on the efficiency of gene delivery. Shuffling of AAV capsid domains may have clinical applications for patients who develop T-cell immune responses following AAV gene therapy, as specific peptide antigen sequences could be substituted using this technique prior to vector re-treatments.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0060361PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3621895PMC
October 2013

Evolution and functional characterisation of melanopsins in a deep-sea chimaera (elephant shark, Callorhinchus milii).

PLoS One 2012 14;7(12):e51276. Epub 2012 Dec 14.

School of Animal Biology, University of Western Australia Oceans Institute and Lions Eye Institute, University of Western Australia, Perth, Western Australia, Australia.

Non-visual photoreception in mammals is primarily mediated by two splice variants that derive from a single melanopsin (OPN4M) gene, whose expression is restricted to a subset of retinal ganglion cells. Physiologically, this sensory system regulates the photoentrainment of many biological rhythms, such as sleep via the melatonin endocrine system and pupil constriction. By contrast, melanopsin exists as two distinct lineages in non-mammals, opn4m and opn4x, and is broadly expressed in a wide range of tissue types, including the eye, brain, pineal gland and skin. Despite these findings, the evolution and function of melanopsin in early vertebrates are largely unknown. We, therefore, investigated the complement of opn4 classes present in the genome of a model deep-sea cartilaginous species, the elephant shark (Callorhinchus milii), as a representative vertebrate that resides at the base of the gnathostome (jawed vertebrate) lineage. We reveal that three melanopsin genes, opn4m1, opn4m2 and opn4x, are expressed in multiple tissues of the elephant shark. The two opn4m genes are likely to have arisen as a result of a lineage-specific duplication, whereas "long" and "short" splice variants are generated from a single opn4x gene. By using a heterologous expression system, we suggest that these genes encode functional photopigments that exhibit both "invertebrate-like" bistable and classical "vertebrate-like" monostable biochemical characteristics. We discuss the evolution and function of these melanopsin pigments within the context of the diverse photic and ecological environments inhabited by this chimaerid holocephalan, as well as the origin of non-visual sensory systems in early vertebrates.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0051276PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3522658PMC
June 2013

Next-generation sequencing (NGS) as a diagnostic tool for retinal degeneration reveals a much higher detection rate in early-onset disease.

Eur J Hum Genet 2013 Mar 12;21(3):274-80. Epub 2012 Sep 12.

Oxford Biomedical Research Centre, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK.

Inherited retinal degeneration (IRD) is a common cause of visual impairment (prevalence ∼1/3500). There is considerable phenotype and genotype heterogeneity, making a specific diagnosis very difficult without molecular testing. We investigated targeted capture combined with next-generation sequencing using Nimblegen 12plex arrays and the Roche 454 sequencing platform to explore its potential for clinical diagnostics in two common types of IRD, retinitis pigmentosa and cone-rod dystrophy. 50 patients (36 unknowns and 14 positive controls) were screened, and pathogenic mutations were identified in 25% of patients in the unknown, with 53% in the early-onset cases. All patients with new mutations detected had an age of onset <21 years and 44% had a family history. Thirty-one percent of mutations detected were novel. A de novo mutation in rhodopsin was identified in one early-onset case without a family history. Bioinformatic pipelines were developed to identify likely pathogenic mutations and stringent criteria were used for assignment of pathogenicity. Analysis of sequencing metrics revealed significant variability in capture efficiency and depth of coverage. We conclude that targeted capture and next-generation sequencing are likely to be very useful in a diagnostic setting, but patients with earlier onset of disease are more likely to benefit from using this strategy. The mutation-detection rate suggests that many patients are likely to have mutations in novel genes.
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http://dx.doi.org/10.1038/ejhg.2012.172DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3573204PMC
March 2013

Melanopsin phototransduction: slowly emerging from the dark.

Prog Brain Res 2012 ;199:19-40

Nuffield Department of Clinical Neuroscience, Nuffield Laboratory of Ophthalmology, John Radcliffe Hospital, Headley Way, Oxford, United Kingdom. Electronic address:

Melanopsin expressing retinal ganglion cells represent a third class of ocular photoreceptors and are involved in irradiance detection and non-image-forming responses to light including pupil constriction, circadian entrainment, and regulation of sleep. Over recent years, there has been a rapid increase in our understanding of the anatomical variety of pRGC subtypes, the regions of the brain which they innervate, and the behavioral responses of melanopsin-based light detection. However, by contrast, our understanding of the intracellular signaling cascade initiated following activation of melanopsin has, until recently, remained poorly characterized. This chapter focus on the melanopsin signaling pathway, detailing the cellular mechanisms of phototransduction that occur within pRGCs, highlighting recent advances, but also the gaps in our understanding of this important light detecting system.
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http://dx.doi.org/10.1016/B978-0-444-59427-3.00002-2DOI Listing
January 2013

Next-generation sequencing in health-care delivery: lessons from the functional analysis of rhodopsin.

Genet Med 2012 Nov 12;14(11):891-9. Epub 2012 Jul 12.

Nuffield Department of Clinical Neuroscience, Nuffield Laboratory of Ophthalmology, John Radcliffe Hospital, University of Oxford, UK.

Purpose: The interpretation of genetic information has always been challenging, but next-generation sequencing produces data on such a vast scale that many more variants of uncertain pathogenicity will be found. We exemplify this issue with reference to human rhodopsin, in which pathogenic mutations can lead to autosomal dominant retinitis pigmentosa.

Methods: Rhodopsin variants, with unknown pathogenicity, were found in patients by next-generation and Sanger sequencing and a multidisciplinary approach was used to determine their functional significance.

Results: Four variants in rhodopsin were identified: F45L, P53R, R69H, and M39R, with the latter two substitutions being novel. We investigated the cellular transport and photopigment function of all four human substitutions and found that the F45L and R69H variants behave like wild-type and are highly unlikely to be pathogenic. By contrast, P53R (a de novo change) and M39R were retained in the endoplasmic reticulum with significantly reduced functionality and are clearly pathogenic.

Conclusion: Potential pathogenicity of variants requires careful assessment using clinical, genetic, and functional data. We suggest that a multidisciplinary pathway of assessment, using several functional assays, will be required if next-generation sequencing is to be used effectively, reliably, and safely in the clinical environment.
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http://dx.doi.org/10.1038/gim.2012.73DOI Listing
November 2012

Differential expression of melanopsin isoforms Opn4L and Opn4S during postnatal development of the mouse retina.

PLoS One 2012 5;7(4):e34531. Epub 2012 Apr 5.

Nuffield Laboratory of Ophthalmology, University of Oxford, Oxford, United Kingdom.

Photosensitive retinal ganglion cells (pRGCs) respond to light from birth and represent the earliest known light detection system to develop in the mouse retina. A number of morphologically and functionally distinct subtypes of pRGCs have been described in the adult retina, and have been linked to different physiological roles. We have previously identified two distinct isoforms of mouse melanopsin, Opn4L and Opn4S, which are generated by alternate splicing of the Opn4 locus. These isoforms are differentially expressed in pRGC subtypes of the adult mouse retina, with both Opn4L and Opn4S detected in M1 type pRGCs, and only Opn4L detected in M2 type pRGCs. Here we investigate the developmental expression of Opn4L and Opn4S and show a differential profile of expression during postnatal development. Opn4S mRNA is detected at relatively constant levels throughout postnatal development, with levels of Opn4S protein showing a marked increase between P0 and P3, and then increasing progressively over time until adult levels are reached by P10. By contrast, levels of Opn4L mRNA and protein are low at birth and show a marked increase at P14 and P30 compared to earlier time points. We suggest that these differing profiles of expression are associated with the functional maturation of M1 and M2 subtypes of pRGCs. Based upon our data, Opn4S expressing M1 type pRGCs mature first and are the dominant pRGC subtype in the neonate retina, whereas increased expression of Opn4L and the maturation of M2 type pRGCs occurs later, between P10 and P14, at a similar time to the maturation of rod and cone photoreceptors. We suggest that the distinct functions associated with these cell types will develop at different times during postnatal development.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0034531PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3320640PMC
August 2012

Anion sensitivity and spectral tuning of middle- and long-wavelength-sensitive (MWS/LWS) visual pigments.

Cell Mol Life Sci 2012 Jul 15;69(14):2455-64. Epub 2012 Feb 15.

UCL Institute of Ophthalmology, London, UK.

The long-wavelength-sensitive (LWS) opsins form one of four classes of vertebrate cone visual pigment and exhibit peak spectral sensitivities (λ(max)) that generally range from 525 to 560 nm for rhodopsin/vitamin-A(1) photopigments. Unique amongst the opsin classes, many LWS pigments show anion sensitivity through the interaction of chloride ions with a histidine residue at site 197 (H197) to give a long-wavelength spectral shift in peak sensitivity. Although it has been shown that amino acid substitutions at five sites (180, 197, 277, 285 and 308) are useful in predicting the λ(max) values of the LWS pigment class, some species, such as the elephant shark and most marine mammals, express LWS opsins that possess λ(max) values that are not consistent with this 'five-site' rule, indicating that other interactions may be involved. This study has taken advantage of the natural mutation at the chloride-binding site in the mouse LWS pigment. Through the use of a number of mutant pigments generated by site-directed mutagenesis, a new model has been formulated that takes into account the role of charge and steric properties of the side chains of residues at sites 197 and 308 in the function of the chloride-binding site in determining the peak sensitivity of LWS photopigments.
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http://dx.doi.org/10.1007/s00018-012-0934-4DOI Listing
July 2012

Rapid assessment of sleep-wake behavior in mice.

J Biol Rhythms 2012 Feb;27(1):48-58

Nuffield Laboratory of Ophthalmology, University of Oxford, John Radcliffe Hospital, Headley Way, Oxford, United Kingdom.

Sleep is a fundamental biological rhythm involving the interaction of numerous brain structures and diverse neurotransmitter systems. The primary measures used to define sleep are the electroencephalogram (EEG) and electromyogram (EMG). However, EEG-based methods are often unsuitable for use in high-throughput screens as they are time-intensive and involve invasive surgery. As such, the dissection of sleep mechanisms and the discovery of novel drugs that modulate sleep would benefit greatly from further development of rapid behavioral assays to assess sleep in animal models. Here is described an automated noninvasive approach to evaluate sleep duration, latency, and fragmentation using video tracking of mice in their home cage. This approach provides a high correlation with EEG/EMG measures under both baseline conditions and following administration of pharmacological agents. Moreover, the dose-dependent effects of sedatives, stimulants, and light can be readily detected. This approach is robust yet relatively inexpensive to implement and can be easily incorporated into ongoing screening programs to provide a powerful first-pass screen for assessing sleep and allied behaviors.
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http://dx.doi.org/10.1177/0748730411431550DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4650254PMC
February 2012

Profound defects in pupillary responses to light in TRPM-channel null mice: a role for TRPM channels in non-image-forming photoreception.

Eur J Neurosci 2012 Jan;35(1):34-43

TRPM1 is a spontaneously active non-selective cation channel that has recently been shown to play an important role in the depolarizing light responses of ON bipolar cells. Consistent with this role, mutations in the TRPM1 gene have been identified as a principal cause of congenital stationary night blindness. However, previous microarray studies have shown that Trpm1 and Trpm3 are acutely regulated by light in the eyes of mice lacking rods and cones (rd/rd cl), a finding consistent with a role in non-image-forming photoreception. In this study we show that pupillary light responses are significantly attenuated in both Trpm1(-/-) and Trpm3(-/-) animals. Trpm1(-/-) mice exhibit a profound deficit in the pupillary response that is far in excess of that observed in mice lacking rods and cones (rd/rd cl) or melanopsin, and cannot be explained by defects in bipolar cell function alone. Immunolocalization studies suggest that TRPM1 is expressed in ON bipolar cells and also a subset of cells in the ganglion cell layer, including melanopsin-expressing photosensitive retinal ganglion cells (pRGCs). We conclude that, in addition to its role in bipolar cell signalling, TRPM1 is involved in non-image-forming responses to light and may perform a functional role within pRGCs. By contrast, TRPM3(-/-) mice display a more subtle pupillary phenotype with attenuated responses under bright light and dim light conditions. Expression of TRPM3 is detected in Muller cells and the ciliary body but is absent from pRGCs, and thus our data support an indirect role for TRPM3 in pupillary light responses.
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http://dx.doi.org/10.1111/j.1460-9568.2011.07944.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4306442PMC
January 2012

Tetradecanoylphorbol-13-acetate (TPA) significantly increases AAV2/5 transduction of human neuronal cells in vitro.

Exp Eye Res 2012 Apr 16;97(1):148-53. Epub 2011 Dec 16.

Recombinant adeno-associated virus type 2 (AAV2) vectors have shown great promise in current ophthalmology clinical trials targeting gene delivery to the retinal pigment epithelium (RPE). To treat the majority of retinal diseases, however, gene delivery would need to be targeted to photoreceptor neurons of the outer retina. AAV2 pseudotyped with the AAV5 capsid (AAV2/5) has shown far greater transduction efficiency in photoreceptors compared to standard AAV2 vectors. For clinical trial applications using gene therapy, it is helpful to generate pre-clinical data in human cells wherever possible. There is however very little data, indeed some controversy, as to whether AAV2/5 can be used effectively in differentiated neurons in culture. In this study we show that transduction of the human neuroblastoma cell line SH-SY5Y with recombinant AAV2/5 expressing GFP is well tolerated. Furthermore, we explore the mechanism whereby exposure to retinoic acid (RA) and the phorbol ester 12-O-Tetradecanoylphorbol-13- acetate (TPA) can induce this cell line to differentiate into a stable population of human neurons, with significantly increased levels of AAV2/5 transduction. These observations may be helpful for assessing AAV2/5 vectors in vitro, particularly where it is necessary to generate pre-clinical data for clinical trials of gene therapy to the human central nervous system.
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http://dx.doi.org/10.1016/j.exer.2011.12.009DOI Listing
April 2012