Optical control of endogenous receptors and cellular excitability with light
Aida Garrido, Nanoprobes and nanoswitches group
Light-controlled manipulation of neuronal activity has transformed the field of neurobiology. Light provides temporal and spatial resolution over activation or inhibition of targeted populations of neurons, one single neuron or single synapses. Such precision could be achieved with optogenetics, which is based on the over-expression of light-sensitive proteins, but it requires gene therapy and often alters cellular physiology. An alternative to optogenetics is offered by photopharmacology (the development of light-regulated drugs or photoswitches), which can operate on endogenous receptors without genetic manipulation. Several new photoswitches are described in this thesis to improve their pharmacological and optical properties.
In photopharmacology, azobenzene is the most commonly used light switch. Photoswitchable tethered ligands (PTLs) are tri-modular molecules able to anchor to target receptors and alter their function by switching the azobenzene group. In the first chapter we demonstrate the ability to target endogenous receptors of hippocampal neurons and olfactory bulb neurons from Xenopus larvae. The chemical strategy used was to introduce a highly reactive anchoring group to a PTL with similar structure to the reported MAG compound used in the light regulated glutamate receptor, LiGluR. These new kind of PTLs were called “Targeted Covalent Photoswitches” (TCPs).
The promiscuity of the reactive group of TCP limits the possibility to spatially confine the drug conjugation. Thus, we designed a photolabile TCP that can be conjugated to its target with a specific wavelength, and then be photoswitched at another wavelength.
Despite the advantages of all molecules described above, they share a common limitation: the activation wavelength of azobenzene is in the UV-violet range. Increasing the push-pull effect at the azobenzenic core by adding different substituents, we achieved a TCP derivative activatable at the visible range and ultrafast relaxing. In this way a single wavelength of stimulation can be used. Both features make them great candidates to control ultrafast neurotransmission processes such as the ones implicated in sound coding in the cochlea. We demonstrated in a gerbil animal model the capacity of this blue-activated TCP to photosensitize native receptors of adult gerbil cochlea. This first proof of concept opens new possibilities to develop optical cochlear implants for the treatment of hearing loss avoiding gene therapy.
Chemical substitutions can also be rationally designed to enhance two-photon (2P) absorptivity without modifying the dynamics of photoswitches. In the third chapter we described two new 2P enhanced MAG derivatives to photocontrol LiGluR. We validated their properties with an all-optical approach, by recording calcium induced neuronal responses in organotypic hippocampal slices, and in vivo in Caenorhabditis elegans. The combination of slow relaxation lifetime and enhanced 2P absorptivity is translated into an accumulation of the active isomer of the photoswitch that successfully enhances its functional effect even at low illumination power.
Another way to spectrally modify the characteristics of a chromophore while avoiding mutation screening and new synthetic processes is by using light-harvesting fluorophores. Spectral overlapping of fluorophore emission and chromophore absorption broadens its activation spectrum. By means of chemical protection and deprotection, we orthogonally control the conjugation of fluorophores and photoswitches. This is the first demonstration of light-harvesting strategy for optogenetics and photopharmacology.
Finally, we describe the use of novel PCLs containing chromophores other than linear azobenzene for the light-controlled activation of hippocampal neurons. Cyclic azobenzenes reverse isomer thermal stability and consequently also the activity of the photoswitch. On the other hand, stilbenes allow the irreversible but non-photo-destructive activation of the original molecule, thus avoiding the creation of photo-products.
In conclusion, this thesis puts forward several advances in the photochromism and pharmacology of photoswitches for the control of neurotransmission without need of genetic manipulation.