Optogenetic methods have the potential to restore the function of photoreceptors and other diseased cell types in the retina. A team from the CIN cluster of excellence at Tübingen University is exploring the basic mechanisms of how this could be achieved.
Degenerative eye diseases like retinitis pigmentosa are associated with the gradual death of the photoreceptor cells in the retina, thereby leading to severe visual impairment and often blindness. Usually, the rod photoreceptors, which are responsible for night vision, are affected first, thereby leading to the loss of night vision. The eventual loss of the cone photoreceptors leads to the loss of daytime vision. It might be decades from the appearance of the first symptoms to total loss of vision, which gives doctors some scope for therapeutic intervention.
Optogenetics seems to be an interesting option for treating retinitis pigmentosa and similar diseases. The principal goal is to restore vision by introducing light-sensitive proteins into the photoreceptors that have lost their function, but are not yet completely dead. A team of researchers at the Werner Reichardt Centre for Integrative Neuroscience (CIN) at the University of Tübingen is studying how this can be achieved and how effective it is.
“In our approach, we are not interested in the mutation that has caused a particular disease,” said Münch, head of the research group Retinal Circuits and Optogenetics. Münch’s team plans to introduce an optogene into non-functional retinal cells using standard virus shuttles. This gene contains the genetic information required for the production of a new channelrhodopsin. Channelrhodopsins, which are related to the biological pigment in the photoreceptor cells of the human retina, are transmembrane proteins that form a channel through which ions can pass. The channel protein opens in response to light: when the channelrhodopsin absorbs a photon, it induces a conformational change in the protein, opening the channel. Ions pass through and trigger a series of reactions that eventually lead to an electrical signal that is passed on to the visual centre where it creates a visual expression.
However, photoreceptors are not the only cells that can be targeted with optogenetic approaches. Optogenes can also be introduced into cells downstream of the retina, especially bipolar cells. This way of regenerating vision has the potential to be used in an advanced stage of disease where patients have lost all cones and rods. Münch’s team has already used mice to study how signal processing changes. “The activation of bipolar cells in an otherwise blind retina leads to signal processing similar to that triggered by a natural response to light,” said Münch referring to his group’s latest findings. He is also considering the possibility of introducing optogenes into ganglion cells, neurons that are even further downstream in the reaction chain than biopolar cells and that transmit visual information to the visual centre in the brain.
The generation and transmission of signals in the retina is one of the issues Münch’s group is investigating. So far very little is known about the detailed visual information processing of the human retina. “Our goal is to expand our basic knowledge about the function of the human retina. In order to do this, we usually employ electrophysiological methods that involve the use of multielectrode arrays,” said Münch. MEAs can be used to analyse the electrical signals of living cells on the tissue and single cell level.
“We have developed a method that enables us to maintain the function of healthy human retinal tissue over several days,” said Münch highlighting that suitable tissue cultures are required for their research. The tissue originates from tumour patients who had to undergo an enucleation due to a tumour and risk of metastasis. “Patients’ willingness to donate an eye is relatively high. However, enucleations are only carried out rarely, which is why we usually only get retinal tissue from our cooperation partners at the University Eye Hospital in Tübingen once a month. Katia Reinhard, a PhD student in Münch’s group, was awarded the 2013 LUSH Prize in the “Young researchers” category, which gave her £12,500 for her research into visual impairment and blindness using human retinal tissue in vitro. The money is now being used to optimise the method. The LUSH Prize is a major initiative that funds research into methods that reduce the use of animals in safety testing.
In addition to such experimental studies, Münch’s group also carries out theoretical studies: “We are using computer models to study how the properties of channelrhodopsin can be altered biophysically so as to make it more sensitive to light,” said Münch. Münch and his master’s degree student Marion Mutter have recently published a study in the scientific journal PLOS ONE that shows that changing specific channelrhodopsin properties allow optogenetic vision over a wider brightness range (spanning two orders of magnitude) than is currently possible – at least in theory. The researchers now have to find out how the sequence of the optogene needs to be modified in order to achieve this improvement. Münch comments: “Although optogenetic stimulation is able to activate cells over a range of light intensities of two orders of magnitude, we are still far from the range of natural vision that can operate over a brightness range of 12 orders of magnitude. The healthy eye easily adapts to different intensities.”
“We know that the light sensitivity of the treated tissue is significantly higher when the channel protein alters its conformation for a longer period of time. More ions can flow through,” said Münch. However, when the channels are open for too long, switching on and off light has no effect. And this is no good either. “We need to concentrate on finding a way to ensure that the conformational changes happen within the time constant in which vision occurs. We use our computer model to represent the individual steps of conformational change using differential equations. We alter the parameters of the equations and carry out computer simulations to find out whether the molecule can be biologically modified,” said Münch. The predictions then need to be tested experimentally. Whatever the outcome, there is nevertheless still a lot of work to be done before specifically modified optogenes can be used in medical applications.
Further information:University of TübingenCIN Werner Reichardt Centre for Integrative NeuroscienceDr. Thomas MünchOtfried-Müller-Str. 2572076 TübingenTel.: +49 (0)7071 29-89182E-mail: thomas.muench(at)cin.uni-tuebingen.de