OPTOGENETICS: USING LIGHT TO MANIPULATE CELL BEHAVIOR IN LIVING ANIMALS.

What is optogenetics?

Optogenetics is a technique that allows to investigate on specific cell processes by precisely manipulating the activity of light-sensitive proteins.

A very widely diffused optogenetic tools is Channelrhodopsin (ChR). ChR was first identified in the early 80s in algae with the ability to respond to light. ChR are light-gated ion channels: they are proteins inserted in the plasma membrane that upon activation with blue light undergo a conformational change that opens the ion channel. Once activated, ChR allows the flow of ions (cations) across the plasma membrane that otherwise would be impermeable to ions or any other charged molecules. The effect of the light on the ion channel is reversible and after switching off the light the channel closes again.

The DNA coding for these light-sensitive proteins can be introduced in cell cultures. For instance, ChR can be expressed in neuronal cultures and, by controlling ion flow, ChR can control the membrane potential and so neuronal excitability. This allows a very precise modulation of the cell behavior, just by modulating the frequency of switching on and off the light. Moreover, different variants of ChR can be obtained by introducing few mutations that can affect for instance the type of light required for the activation.

ChR is just one example of the optogenetic tools today available.

For instance, optogenetic tools that allow to manipulate the synthesis of second messengers such as cAMP are becoming more and more popular. These proteins have a light-sensitive domain and by switching on and off blue light (see glossary) is possible to control the synthesis of cAMP. More recently, it has been developed a light-sensitive phosphodiesterase (PDE). PDEs are the enzymes responsible for the degradation of cAMP. By shining red light, these light-sensitive PDEs are activated, resulting into cAMP degradation, while shining far red light switches off the enzyme, stopping cAMP degradation. Using the light to modulate cAMP concentration allows to finely control the related cell processes, such as axon outgrowth or cell migration.

Due to the possibility of controlling these proteins easily and precisely, these tools appear valuable for in vivo purpose.

Luckily technology and science progress proceeds in parallel, and today companies offer different ways to modulate these tools in vivo with light. Optical fibers can be implanted in mice in the specific tissue expressing the optogenetic tools. Light stimulation is controlled from the outside with technical devices delivering light at specific wavelength, specific frequency and enabling to control also other important parameters. First approaches have used ChR in epilepsy, Parkinson’s Disease or spinal cord injury.

How can we use optogenetics?

Optogenetics is widely used in retinal diseases. Using light-controlled proteins to modulate cell physiology is indeed easier in the retina that is already exposed to light. In this case, there is no need of surgery and optic fibers implantations.

The retina is a highy organized structure, made of different layers: it is delimited on the apical side by the retinal pigmented epithelium (RPE), a single layer of epithelial cells. Photoreceptors are adjacent to the RPE and make synapses with bipolar neurons that, in turn, are linked to retinal ganglion cells. The axons of retinal ganglion cells gather together making the optic nerve. Photoreceptors are responsible for light perception: they translate the light signal into a chemical signal that is then transmitted through the different retinal layers, to the optic nerve that conveys the information to the brain to be processed.

Optogenetics has been used to treat Retinitis Pigmentosa (RP). RP is a degenerative disease causing photoreceptors death. Losing the cells responsible for light perception results into blindness (the severity of blindness depends on the percentage of photoreceptors lost. Usually, the peripheral retina is affected first, resulting in what is commonly called “tunnel vision”. Eventually, this is followed by the loss of central vision and complete blindness).

Studies have shown that in these patients about 80% of the bipolar cells are preserved even if the photoreceptors are mostly gone. Expressing ChR in bipolar cells enables the cells to replace photoreceptors in light perception.

In order to selectively target bipolar cells over other retinal population, ChR is expressed in vivo with viral vectors (see glossary). The vectors can be injected in the eye with an easy procedure that is today routinely performed in patients. Recording the electrical activity of retinas ex vivo (see glossary) showed that ChR expression renders bipolar cells light-sensitive. So, even in absence of photoreceptors, light signal is now perceived by bipolar cells -as a change in their excitability- and conveyed from bipolar cells, through retinal ganglion cells, to the brain.

Experiments have shown that with this approach mice affected by RP (so mice that have lost their photoreceptors) recover light sensitivity. Indeed, when RP mice expressing ChR in their retinas were placed in a transparent cylinder and stimulated by switching on blue light, they promptly reacted to the light, very similarly to healthy mice, while untreated RP mice slowly reacted to the light.

Additional studies are required to check what is the actual visual recovery of the mice with this approach. However, the results are very encouraging because now we know that is actually feasible to introduce an exogenous gene in living animals, and restore their light-sensitivity with such an easy technique, an aspect that researchers cannot forget when thinking of a future biomedical translation of their studies. However, a light-sensitivity test does not say much about the actual benefit of the approach to restore vision and the ability to process the image. The high specificity in targeting the different cell subtypes in the retina, perhaps ChR with different light sensitivities, combined with a thorough analysis of the contribution of each cell type in the retinal circuitry, necessary for decoding the visual information, will definitely be key to achieve a proper treatment. However, it seems that we are going to the right direction. We just need to refine our tools in order to make everything more effective. Does this seem science fiction? Maybe, but so optogenetics seemed only few years ago.

Glossary:

Viral vectors. Tools used to insert specific DNA in cells, by taking advantage of the ability of viruses to transport their DNA into the cells they want to infect. The introduction of DNA into a cell is called “transduction”. The DNA that we want to express is packaged into the “protein envelop” of a virus. Even if they are derived from viruses, they are modified in order to be non-infective. They can be engineered to transduce only one specific cell type.

Blue light. Light is defined as a wave, with all the specific properties of a wave, such as for instance the wavelength. In a wave whose shape repeats –eg a sinusoid- the wavelength is defined as the distance between two crests. Waves of different wavelengths are perceived by the eye/brain as different colors: for instance, blue light has a wavelength of approximately 480nm, while red light is around 600nm.

In vitro, in vivo, ex vivo. An experiment is “in vitro” when is performed with cells in a dish, “in vivo” when is performed in living animals, “ex vivo” when is performed in tissue directly isolated from a living animal. Just to be precise, some PDE isoforms catalyze the hydrolysis of both cAMP and cGMP. So, light activation of this PDE triggers degradation of both second messengers.

References:

Targeting channelrhodopsin-2 to ON-bipolar cells with vitreally administered AAV Restores ON and OFF visual responses in blind mice. Macé, Caplette, Marre, Sengupta, Chaffiol, Barbe, Desrosiers, Bamberg, Sahel, Picaud, Duebel, Dalkara. Molecular Therapy 2015.

Channelrhodopsins: visual regeneration and neural activation by a light switch. Tan, Farhatnia, Rajadas, Hamblin, Khaw, Seifalian. N Biotechnol. 2013

Retinal stimulation strategies to restore vision: Fundamentals and systems. Yue, Weiland, Roska, Humayun. Prog Retin Eye Res. 2016

Engineering of a red-light-activated human cAMP/cGMP-specific phosphodiesterase. Gasser, Taiber, Yeh, Wittig, Hegemann, Ryu, Wunder, Möglich. PNAS 2014.