Photochemical control of neuronal activity: methods and clinical application

Photochemical control of neuronal activity: methods and clinical application/nby Ivan Tochitsky/nDoctor of Philosophy in Molecular and Cell Biology/nUniversity of California, Berkeley/nProfessor Richard Kramer, Chair/nMammalian nervous systems are incredibly complex, with almost 100 billion neurons making up the human brain. Neurons in the brain primarily communicate with one another in one of two ways - electrically, via the flow of ions across the cell membrane, or chemically by releasing and detecting a variety of signaling molecules. In order to understand the function of the nervous system, we need to be able to manipulate it with high spatial and temporal precision. Conventional electrical or chemical stimuli do not allow for such precise control. Thus, a new and orthogonal stimulus modality had to be utilized in order to facilitate the study of the nervous system. The emerging field of optogenetics uses light as such a stimulus since light can be delivered only to a small part of the nervous system, or even a single neuron, and the illumination can be controlled with millisecond time resolution. Optogenetic techniques involve the expression of light-sensitive proteins from microbes in genetically targeted populations of neurons, rendering those neurons sensitive to light. Recent advances in optogenetics have greatly advanced our understanding of the function of the nervous system both in healthy organisms, and in the context of disease. Optogenetics is a powerful technique for investigating neural networks, but this approach primarily studies the function of the nervous system at a system rather than molecular level. The vast complexity of the human brain is created not only by the large number of individual neurons and the intricate connections between them, but also by the dizzying variety of proteins found in the cell membranes of these neurons. These proteins sense and respond to the release of chemical signaling molecules from neighboring cells, or changes in ion concentrations that alter the cell's membrane potential, allowing for the generation and propagation of electrical signals./nWe have combined the powers of synthetic chemistry and genetics to develop novel optopharmacological or optochemical genetic methods which enable precise optical control of neuronal function at the molecular level. These strategies involves the generation of light-sensitive "photoswitch" molecules that selectively target a population of either genetically engineered or endogenous membrane proteins - including receptors sensing chemical stimuli, or ion channels responding to electrical potential changes in the cell. The addition of a photoswitch compound to a neuron expressing the target protein makes that protein, and, by extension, the neuron, sensitive to light. We first applied this strategy to generate light regulated neuronal nicotinic acetylcholine receptors, which are a group of proteins that respond to the chemical neurotransmitter acetylcholine. These receptors modulate the activity of other neurons in different parts of the brain and are also sensitive to nicotine, an addictive chemical found in tobacco products. The function of acetylcholine receptors in the brain and their role in nicotine addiction, neuropsychiatric and neurodegenerative disorders is not fully understood, in large part because it quite difficult to chemically manipulate individual receptors without affecting others. Making light-sensitive, genetically targeted acetylcholine receptors should thus greatly advance our understanding of those receptors' function./nThe main rationale for making proteins or neurons light-sensitive is to facilitate the study of the healthy nervous system as well as its malfunction in disease. There are, however, several human diseases where optical methods for controlling neuronal activity could directly provide a clinical benefit. Degenerative blinding diseases such as retinitis pigmentosa or age-related macular degeneration leave the retinas of affected patients either partly or completely insensitive to light by causing the death of light-detecting photoreceptor cells in the eye. Light responses can be restored to a blind retina by making some or all of the remaining retinal neurons sensitive to light. This can be achieved via the expression of light sensitive microbial opsins or engineered receptors in retinal neurons that are not normally light sensitive. Both of these approaches have restored some visual perception to blind mice suffering from retinitis pigmentosa. However, in order to use either optogenetic or optochemical genetic tools in the clinic, the mutant proteins must be artificially expressed in the patient's retina, which requires the use of viral gene therapy. Gene therapy has potential health risks, so we decided to develop a treatment for blinding diseases that would only involve a light-sensitive chemical, without the need for gene therapy or invasive surgery./nTo that end, we have developed an optopharmacological therapy for vision restoration by creating photoswitch molecules that block and unblock endogenous voltage-gated ion channels in a light-dependent manner, allowing us to control almost any neuron with light. The first photoswitch tested, called AAQ, restored electrical retinal light responses, the pupillary light reflex, as well as other simple visual behaviors in blind mice. In order to optimize this treatment for clinical use, we generated a compound called DENAQ with improved light sensitivity and persistence in the eye, which responds to broad spectrum white light, similar to what people encounter in natural visual scenes. Furthermore, DENAQ acts selectively on retinas suffering from photoreceptor cell death, but leaves healthy retinas unaffected. This selectivity raises the possibility that we may be able to treat not only patients who are completely blind, but also those suffering from partial vision loss, by restoring light sensitivity only to the parts of the retina experiencing photoreceptor degeneration. The promising preliminary results from animal studies suggest that our optopharmacological strategy for vision restoration may eventually be used in the clinic, in addition to helping researchers understand the function of the nervous system in its normal state and in disease.

Advisor: Kramer, Richard H