Functional domains in melanopsin: natural variants and molecular engineering

Rods and cones are light sensitive cells in the retina responsible for dim light vision and colour vision. More recently a third type of light sensitive cell has been found in the retina, which express the blue-light sensitive protein called melanopsin. These cells, termed photosensitive retinal ganglion cells (pRGCs), send connections to numerous structures within the brain and control a range of non visual responses to light including the induction of sleep, pupil light reflexes and entrainment to daily light cycles. More recently these cells have been shown to perform roles in visual pathways, mood and depression, and are emerging as a key biological pathway regulating human physiology and health.

Mutations in the light sensitive molecules of rods and cones lead to a range of retinal diseases and are a leading cause of blindness. Mutation and polymorphisms in melanopsin have also been detected in the human population, resulting in changes in physiological responses to light and have been associated with seasonal affective disorder. Understanding the consequences of mutations on the properties of melanopsin is fundamental to predicting their effects on human physiology and health. As with all biological molecules, the structure of melanopsin is key to its function. However the functional roles performed by the different regions of the melanopsin molecule are not well established.

In this study we will produce a number of melanopsin molecules containing targeted changes within specific regions of the protein, as a means to determining the roles performed by these regions in a number of key functions. Principally we will focus on modelling the mutations known to be present in the human population, and determine the general roles performed by areas of the molecule in which these mutations are found. In addition, we will determine the regions of the melanopsin protein that influence sensitivity to light and specifically the colour of light to which this protein responds (termed spectral tuning sites). Similar studies have been conducted for the light sensitive 'opsin' molecules found in rods and cones, yet melanopsin represents a functionally different class of opsin with important functional and structural differences. To date no study has investigated the spectral tuning of this class of 'non-visual' opsin. As a consequence of this study we will generate a range of melanopsin variants that respond to different colours of light. Such variants have huge potential for research applications, where the expression of melanopsin can be used to confer sensitivity to light and allow the selective activation of cell types of interest both in vitro and in vivo, and also for gene therapy based approaches aimed at treating retinal disease and restoring vision.

This programme of work will lead to a fundamental increase in our understanding of the melanopsin protein, a protein that performs important roles in human physiology and health. This study will also provide a range of new research tools that are applicable to a wide range of biological research fields and also the development of novel tools for the treatment of human disease.

Melanopsin expressing photosensitive retinal ganglion cells (pRGCs) represent a third class of photoreceptor and mediate a range of non-image forming responses to light, including circadian entrainment, pupil constriction and the induction of sleep. More recently it has become clear that the circadian system may perform important roles in human health and disease. However, despite the significant advances in our understanding of the anatomy and function of the melanopsin system, there is much that is not known, both concerning the properties of the melanopsin molecule itself, the cell signalling pathways initiated by melanopsin activation, and also how naturally occurring mutations in the melanopsin gene influence the properties of the melanopsin photopigment, the properties of melanopsin driven light responses, and downstream behavioural response to light. This study will use site directed mutagenesis to generate a range of melanopsin variants, including those that replicate single nucleotide polymorphisms (SNPs) identified in the human population, and also variants containing substitutions in key function domains and at spectral tuning sites. Cutting edge AAV viral delivery vectors will be used to selectively express these melanopsin variants within pRGCs of the mouse retina in vivo, providing the optimal native environment to study the functional properties of these pigments. Functional analysis of melanopsin variants at the protein, cellular and behavioural levels will be used to determine the functional effects of these mutations on melanopsin function and identify the functional roles performed by specific domains of the melanopsin protein. Furthermore, this study will conduct the first analysis of spectral tuning sites in any class of bistable opsin, and lead to the generation of a range of spectrally tuned variants of melanopsin that will have significant applications as optogenetic research tools.

The impact of this proposal is expected to extend well beyond the research community, building upon the existing communication networks and expertise of the applicants. Both Dr Peirson (SNP) and Prof Hankins (MWH) have a track record in securing industrial collaborations, and given the relevance of melanopsin to human physiology and health, and the nature of biological tools developed throughout this project, this proposal is expected to lead to further industrial partnerships. Both SNP and MWH have been involved in communicating their research to third sector organisations such as the RNIB, and SNP has also been involved in workshops associated with public policy, such as the NC3Rs, Home Office and Public Health England. Both MWH and SNP have a track record in public understanding of science, contributing to Radio 4 and New Scientist, respectively. MWH is also a member of the BBSRC Bioscience for Society Strategy Panel. Strong research interaction between the Nuffield Laboratory of Ophthalmology (NLO) and the Oxford Eye Hospital (OEH) also enables engagement and involvement of those working in front-line health care, and presentations on the OEH lecture series and the NLO's annual "Updates in Ophthalmology" meeting will enable both specific details and health care implications of this work to be communicated to health care professionals. The applicants have a strong track record of innovation and developing novel research tools and applications. This background provides a working knowledge of the processes required to protect and exploit research findings, which may have applications far beyond the scope of the immediate project. Finally, with the recent developments of the NLO and NDCN websites, the applicants have a further tool to ensure the current proposal achieves the greatest possible impact.