Author: Isabelle Arnoux Edited by: Vanessa Hübner
Optogenetics is a recent technique using light to influence the activity of genetically modified cells. In 2005, a pioneering study lead by Karl Deisseroth introduced this approach (1) and five years later, the interdisciplinary journal Nature Methods rewarded this breakthrough as the “method of the year”. In the field of neuroscience, optogenetics has revolutionized the way to map the contribution of individual neurons on networks and brain circuitries. Targeted neurons are genetically modified to express light-sensitive proteins – optogenetics actuators –, which can turn the activity of a specific neuronal population either on or off.
Opsins convert light into electrochemical signals
The mainly used actuators are opsins, which are classified in three groups: ion channels, pumps and G proteins-coupled receptors. Opsins change their shape upon light absorption from a resting state to an active/signalling state. They convert light into an electrochemical signal to trigger the modulation of intracellular signalling cascades, the opening of pore channels or the activation of pumps. Then, the stimulation of ion channels or pumps induces a flux of ions that leads to a depolarization (activation) or a hyperpolarisation (inhibition) of the membrane potential of the respective neuron. For example, the activation of the blue-light sensitive channelrhodopsin (the most common opsin) increases the neuronal activity, whereas the activation of yellow-light sensitive archaerodopsin reduces it. Notably, the change of the conformation of the opsins is induced by a specific wavelength of light which depends on the internal properties of the opsin
But, opsins are not physiologically expressed in mammalian cells. Consequently, scientists developed strategies to enable their expression in post-mitotic cells. The most popular one is the viral gene transfer, which is easily implementable in laboratories and allows fast and robust expression in targeted cells. They engineered viruses, mostly lenti- and adeno-viruses, that contain the opsin gene driven by a specific promoter. Then, this virus is injected into the region of interest and after an infection period of several weeks, the opsin is expressed at the membrane of the targeted neurons in a spatially restricted area. The choice of the virus type and the promoter is critical to achieve cell type specificity – a fundamental advantage of optogenetics. For example, while the synapsin promoter induces opsin expression in all types of neurons, the CaMKII (calcium/calmodulin dependent protein kinase II) promoter restricts the expression to excitatory glutamatergic neurons, one specific type of neurons. In addition, the virus serotype modulates the tissue specificity and the efficiency of opsin expression: adenovirus 1, 2, 5, 8 and 9 are commonly used to affect neurons with differential efficacies and spatial extent.
Optogenetic approaches in neurological diseases
The specific modulation of neuronal circuit activity through the optogenetic technology holds biomedical promise to treat or attenuate disease progression and symptoms. This method has already been used with success in animal models of disorders of the nervous system, such as Parkinson’s disease, Alzheimer’s disease, spinal cord injuries and retinitis pigmentosa.
Parkinson’s disease is a neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra, a structure of the basal ganglia, which is involved in motor control. Consequently, major symptoms in Parkinson’s disease are tremor, slowness of movement, rigidity and postural instability. Worldwide, 6.5 million people are afflicted with this disease. In 2009 and 2010, two innovating studies applied newly developed optogenetic tools in a mouse model of Parkinson’s disease (2) (3). They identified dysregulated elements in the primary motor cortex and striatum and showed that their optical stimulation reduced motor behavioural deficits. Recently, it has been reported that optical activation or inhibition of two distinct groups of neurons located in the external globus pallidus, a structure of the basal ganglia, restores movement in a mouse model of Parkinson’s disease (4). Indeed, the optical regulation of specific neurons expressing channelrhodopsin (parvalbumin interneurons) or archaerhodopsin (Lhx6 neurons) can improve behaviour deficits for several hours by re-balancing the burst firing of neurons in the substantia nigra reticulata, a down-stream structure in the basal ganglia circuitry. In addition, for the first time, this effect on behaviour persisted for hours and provided the basis for new therapeutic applications, which can be more effective than current therapies.
The use of optogenetics to alleviate symptoms of neurological disorders has also been applied in Alzheimer’s disease, another neurodegenerative disorder characterized by amyloid-beta plaques and intracellular aggregates of tau protein. The symptoms include dementia, loss of memory, problems with language, disorientation and behavioural issues. Currently, 35.6 million of people aged 65 years or more are affected by Alzheimer’s disease and this number will double in 2030. Consequently, finding a therapeutic treatment to treat Alzheimer’s disease is critical for our ageing society. The memory deficit, an early pathological hallmark, could be due to an ineffective encoding of new information (learning and maintaining the information as memory) or a disrupted retrieval of the learned information (remembering). In this context, optogenetics has been used to restore memory by stimulating neurons located in the dentate gyrus, a region of the hippocampus, which plays a role in learning and memory (5). Excitatory opsins expressed by neurons that were previously involved in learning, were then optogenetically activated to retrieve the learned information in a mouse model of Alzheimer’s disease. This indicates that the memory deficit in early Alzheimer’s disease are deficits in the retrieval of information – and that these deficits can be rectified by the stimulation of a group of specific neurons. However, it is still unknown whether correcting the deficit in memory will prevent the onset of late symptoms.
Beyond brain applications, optogenetics could also be used to treat peripheral nervous system disorders: As such, spinal cord injuries result in the loss of connection between the brain and the body. Therefore, treatments that stimulate nerves could restore some motor functions. For decades, researchers focused on molecular treatments aiming to attenuate secondary injuries and to promote axonal regrowth – and so far, have proven to be ineffective. Also, functional electric stimulations to activate neuromuscular units showed various limitations: non-specificity, electrical current spill over, muscle fatigue and only one lost function could be corrected at the time. However, optogenetics could overcome these limitations: First, the novel technique allows motoneurons to selectively express opsins in order to stimulate the targeted muscle fibres precisely. Second, optogenetics enables the activation and inhibition of different neuronal populations with multiple opsins being stimulated by different wavelengths – a condition that is needed for complex functions. Third, with optogenetics, muscle fatigue can be avoided by preferentially activating fatigue-resistant fibres. In 2013, a research group from Stanford performed intramuscular injections of adeno-viruses driving the expression of channelrhodopsin in a population of peripheral motor neurons to innervate the injected muscle (6). To stimulate the nerves, they implanted a light delivery system: a biocompatible optical fibre-based nerve cuff that wraps around the targeted nerve in freely moving rats. Notably, the optical stimulation of the nerve elicited muscle activation that is restricted to the targeted muscle. This method was applied to treat respiratory insufficiency in a rat model of spinal cord injury (7). The optogenetic approach could restore the respiratory motor function by recovering the muscle activity even after the ending of the light pulse. In the long term, optogenetics could possibly be applied to control locomotion functions, which will greatly improve the quality of a patient’s life.
Another disease amenable for optogenetic treatment is the retinitis pigmentosa, which is characterized by the loss of photoreceptors leading to complete blindness. In this case, optogenetics can be applied to restore photosensitivity in bipolar cells of degenerated retinas (8). Indeed, the activation of channelrhodopsin-expressing bipolar cells in a mouse model of the disease is sufficient to induce light-evoked spiking activity in ganglion cells and photo responses are transmitted to the visual cortex. These results were also obtained when inner retinal neurons are optogenetically stimulated. In addition, the optical activation of bipolar cells drastically increased performance in visual behavioural tasks showing an amelioration of vision.
Optogenetics in humans
Until now, only few patients affected by retinitis pigmentosa have been enrolled in clinical trials to undergo optogenetic therapy. In 2016, the first human test was initialized in Texas and sponsored by a start-up called RetroSense Therapeutics. To restore vision, they injected engineered viruses carrying the channelrhodopsin gene in the eye of patients to create new photosensors in retinal cells. Upon natural light penetration, these cells will generate electrical signals that are then conveyed to the visual cortex. However, since opsins only respond to specific wavelengths, natural light might not be bright enough to induce the desired activity. Unfortunately, RetroSense Therapeutics has not communicated their results yet and we still do not know if the optogenetic therapy was effective. Consequently, some new companies emerged developing glasses to improve the vision by optogenetics. Gensight Biologics in Paris designed an external wearable medical device in the form of “biomimetic goggles”, containing a camera, a microprocessor and a digital micromirror array. These goggles process camera recordings of life scenes into bright pulses of light at specific wavelengths to stimulate modified retinal cells. Thus, the retinal activity under normal light conditions is mimicked by amplifying light signals. These googles (“GS030”) will be tested in humans in the next year. Another company, founded last year in New York and called Bionic Sight also developed a technique involving optogenetics and a wearable device.
It is still unknown what sort of vision is generated from the stimulation of modified retinal cells. Since there are diverse types of retinal ganglion cells that are activated by light, colour, contrast and motion, it is likely that a lot of research and development will be needed to ultimately target a specific type of retinal cells and thus, to re-create the signals flowing to the visual cortex to obtain a correct perception of natural scenes.
Beyond the treatment of blindness, this first human trial offers promises of potential therapies for treating patients suffering from different neurological diseases. Circuit Therapeutics, a company based in California, also works on an optogenetic therapy to understand the cause of different neurological diseases, to map disease circuitries and to develop new drugs for treatment.
Optogenetic therapeutical treatment offers many advantages compared to pharmacological and electrical treatments, which are commonly used to treat neurological disorders.
First, in contrast to the unselective treatment on the activity of different cell types of pharmacological and electrical approaches, optogenetics allows the targeted regulation of a specific cell population or circuit.
Second, optogenetics displays a fast response compared to pharmacological treatments. Optical stimulation can affect neuronal activity in a millisecond timescale temporal resolution, whereas drug treatments take several minutes before inducing an effect.
Third, optogenetics could also be safer. While deep brain stimulation requires a risky surgical operation to implant the electrodes in the brain and neurostimulators under the skin to trigger current pulses, the optogenetic treatment requires gene therapy and the implementation of a light device (externally wearable as described for retinitis pigmentosa) but no oscillating electric field that will be pulsed through the body.
However, optogenetic therapy in humans also exhibits some inconveniences:
The first obstacle is to achieve a good expression of opsins in the human brains. Indeed, the human immune system can react against viral transfection methods used for the delivery of genes encoding light-sensitive proteins. The best solution is to proceed the virus injections with adeno-associated viruses or lentiviruses, which have low pathogenicity, low immunogenicity, high efficiency, and a long-lasting transgene expression. These viruses have already been used in the cortex of primates to express a functional channelrhodopsin in neurons and the subsequent histological analysis did not reveal immune reactions or cellular abnormalities after months (9). Furthermore, optogenetic stimulation of cortical neurons from two monkeys during the execution of a visually guided saccade task induced behavioural and functional changes in the neural network. This study not only indicates that opsin expression in primate brains – and due to its closeness to the human brain – may be a potentially safe process, but also that optogenetic neuromodulation therapy is cell-specific in the long term. Nevertheless, the insertion of light-responsive genes into cells includes risks due to the potential of opsin overexpression, phototoxicity and tissue damage caused by the repeated optical stimulation. Consequently, more experimental tests are still required.
Importantly, optogenetic therapy in humans raises ethical concerns. This therapy modifies human gene expression in an irreversible way via introduction of non-human DNA and induces an external regulation of cell activity and the neural network. This external control of brain activity can lead to governed behaviours through motor and psychological actions and imply a third part in the control of human actions and decisions. In this context, some limitations must be set to avoid abuses and misuses of this technique in humans.
1. Millisecond-timescale, genetically targeted optical control of neural activity. Boyden ES, Zhang F, Bamberg E, Nagel G, Deisseroth K. 2005, Nature Neuroscience.
2. Optical deconstruction of parkinsonian neural circuitry. Gradinaru V, Mogri M, Thompson KR, Henderson JM, Deisseroth K. 2009, Science.
3. Regulation of parkinsonian motor behaviours by optogenetic control of basal ganglia circuitry. Kravitz AV, Freeze BS, Parker PR, Kay K, Thwin MT, Deisseroth K, Kreitzer AC. 2010, Nature.
4. Cell-specific pallidal intervention induces long-lasting motor recovery in dopamine-depleted mice. Mastro KJ, Zitelli KT, Willard AM, Leblanc KH, Kravitz AV, Gittis AH. 2017, Nature Neuroscience.
5. Memory retrieval by activating engram cells in mouse models of early Alzheimer's disease. Roy DS, Arons A, Mitchell TI, Pignatelli M, Ryan TJ, Tonegawa S. 2016, Nature.
6. Optogenetic control of targeted peripheral axons in freely moving animals. Towne C, Montgomery KL, Iyer SM, Deisseroth K, Delp SL. 2013, PLoS One.
7. Light-induced rescue of breathing after spinal cord injury. Alilain WJ, Li X, Horn KP, Dhingra R, Dick TE, Herlitze S, Silver J. 2008, Journal of Neuroscience.
8. Light-activated channels targeted to ON bipolar cells restore visual function in retinal degeneration. Lagali PS, Balya D, Awatramani GB, Münch TA, Kim DS, Busskamp V, Cepko CL, Roska B. 2008, Nature Neuroscience.
9. Millisecond-timescale optical control of neural dynamics in the nonhuman primate brain. Han X, Qian X, Bernstein JG, Zhou HH, Franzesi GT, Stern P, Bronson RT, Graybiel AM, Desimone R, Boyden ES. 2009, Neuron.