Two-photon neuronal and astrocytic stimulation with azobenzene-based photoswitches

This is an open access article published under an ACS AuthorChoice License. See Standard ACS AuthorChoice/Editors' Choice Usage Agreement - https://pubs.acs.org/page/policy/authorchoice_termsofuse.htmlSynthetic photochromic compounds can be designed to control a variety of proteins and their biochemical functions in living cells, but the high spatiotemporal precision and tissue penetration of two-photon stimulation have never been investigated in these molecules. Here we demonstrate two-photon excitation of azobenzene-based protein switches and versatile strategies to enhance their photochemical responses. This enables new applications to control the activation of neurons and astrocytes with cellular and subcellular resolution

Photoswitchable glutamate receptors to control neurotransmission with light

[cat] L’estudi de la neurotransmissió requereix noves eines moleculars, i els fotocommutadors ofereixen grans possibilitats. Aquesta tesi està centrada en l’ús de receptors de glutamat activables per llum (LiGluRs) pel control de l’activitat neuronal i dels processos de neurosecreció. Al primer bloc de resultats, la permeabilitat a Ca2+ dels receptors de glutamat s’aprofita per manipular de manera directa -independentment del voltatge de membrana- i reversible la concentració intracel•lular de Ca2+ amb llum. Així, és possible desencadenar els processos de secreció en cèl•lules cromafins i de neurotransmissió en neurones hipocampals. A la segona part dels resultats de la tesi, s’investiga l’estimulacio multifotó del receptor de glutamat modificat químicament amb fotocommutadors basats en l’azobenzè. Els resultats mostren l’estimulació per dos fotons del LiGluR, incloent-hi dos fotocommutadors nous, que milloren l’absorció multifotó del commutador azobenzè. Finalment, s’aplica aquesta tècnica per estimular neurones i astròcits amb una resolució a l’escala d’una cèl•lula o de compartiment subcel•lular. Al tercer capítol dels resultats, es descriu un nou mètode per aconseguir el fotocontrol de receptors neuronals endògens, utilitzant lligands covalents. Amb l’aplicació d’aquest mètode es pot fotocommutar l’activitat del receptor de kainat subtipus 1 (GluK1) no mutant, quan se sobreexpressa el receptor en cèl•lules de mamífer. Aquesta estratègia també permet obtenir fotocorrents en cultius de neurones dels ganglis de l’arrel dorsal, on GluK1 és la subunitat de glutamat endògenament més expressada. Els nous mètodes desenvolupats en aquesta tesi milloren la utilització dels LiGluRs, encaminant l’ús dels fotocommutadors cap al control òptic dels receptors endògens, amb la possibilitat d’estimular cèl•lules individuals o estructures subneuronals, fet que situaria els fotocomutados com a eines indispensables per l’estudi del cervell, des de la fisiologia fins als circuits neuronals.[eng] Optical tools to control neuronal activity include synthetic photoswitchable ligands of receptors and ion channels. Photoswitches can act either as soluble molecules (photochromic ligands, PCLs) or tethered to the protein (photoswitchable tethered ligands, PTLs), and they have been used to photocontrol many ion channels and receptors such as voltage-gated potassium channels, acetylcholine or glutamate receptors. Recognizing both the need for new optical tools in neuroscience and the opportunities offered by photoswitches, this work is focused on the use of light gated glutamate receptors to control neuronal activity and neurotransmission. In the first chapter of results of the thesis, we demonstrate that the Ca2+-permeable LiGluR can be used as a tool to reversibly control neurosecretion by directly affecting the intracellular [Ca2+]. To achieve this goal, LiGluR was expressed in cultured bovine chromaffin cells and cultured hippocampal neurons. We measured secretion in chromaffin cells using two techniques, amperometry and membrane capacitance, and current-clamp recordings to assess neurotransmission in cultured neurons. The results indicated that the magnitude of LiGluR-mediated Ca2+ influx is sufficiently large to trigger regulated exocytosis in chromaffin cells and neurons. In addition, LiGluR induced secretion can be modulated with the wavelength of illumination. This new application of LiGluR opens the possibility to reversibly control the activity of individual synapses, which might help to understand the computational properties of neurons and to unravel how brain circuits work. To use LiGluR as an effective method to interrogate the neuronal function it should support high-spatial 3D resolution and tissue penetration. Multiphoton excitation with near-infrared light enables stimulation in intact tissue with cellular and subcellular resolution, and it has been extensively applied to optical actuators such as caged compounds and more recently to optogenetics. However, two-photon stimulation of synthetic photoswitches had not been explored before. In the second section of the results, the two-photon stimulation of LiGluR is investigated. Two new photoswitches were designed (MAG2p and MAGA2p) based on the structure of the original photoswitch (MAG) and intended to enhance the two-photon absorption ability of the azobenzene switch. The three PTLs, including MAG, successfully activate LiGluR under two-photon stimulation, suggesting that multiphoton excitation can be applied to other azobenzene-based molecules. Interestingly, the rationally designed photoswitches were more efficient in opening LiGluR as lower power and shorter simulation time were required. Finally we validated MAG2p and MAG as new tools to control the activation of neurons and astrocytes with cellular and subcellular resolution. In the last chapter, a new method based on the affinity labeling approach is presented in order to confer light sensitivity to endogenous receptors. Glutamate-azobenzene-reactive PTLs with different lengths and reactive groups were tested on kainate receptors. These reactive PTLs successfully and functionally conjugate to the ionotropic glutamate receptor subunit GluK1, thus enabling to photoswitch its activity, as evidenced from photocurrent recordings of mammalian cells overexpressing the non-mutated receptor. These results are also supported by the photocontrol of GluK1 currents in dorsal root ganglion neurons, where GluK1 is the main glutamate subunit that is endogenously expressed. The new strategy proposed is versatile and we suggest that it can be extended to label other endogenous receptors, giving rise to novel optpharmacological therapies. The new methods here developed improve the LiGluR performance, and address photoswitches to use endogenous neuronal receptors to optically control neuronal activity, being able to stimulate them in small volumes corresponding to the neuronal functional unit (i.e. synaptic terminals). In this way, light would emerge as a unique tool to dissect neuronal physiology and to understand the function of neuronal circuits

Optical control of endogenous receptors and cellular excitability using targeted covalent photoswitches

Light-regulated drugs allow remotely photoswitching biological activity and enable plausible therapies based on small molecules. However, only freely diffusible photochromic ligands have been shown to work directly in endogenous receptors and methods for covalent attachment depend on genetic manipulation. Here we introduce a chemical strategy to covalently conjugate and photoswitch the activity of endogenous proteins and demonstrate its application to the kainate receptor channel GluK1. The approach is based on photoswitchable ligands containing a short-lived, highly reactive anchoring group that is targeted at the protein of interest by ligand affinity. These targeted covalent photoswitches (TCPs) constitute a new class of light-regulated drugs and act as prosthetic molecules that photocontrol the activity of GluK1-expressing neurons, and restore photoresponses in degenerated retina. The modularity of TCPs enables the application to different ligands and opens the way to new therapeutic opportunities.We are grateful to G. Swanson (Northwestern University Feinberg School of Medicine) for the GluK1-2b receptor clone and advice for DRG neuronal culture, and to M. Mayer (National Institutes of Health) for GluK1 S1S2 plasmid and purification protocol. We also thank E. Vázquez (Institut de Biotecnologia i Biomedicina, Universitat Autònoma de Barcelona) for providing the Origami-B (DE3) strain, and O. Seria, J.A. del Rio, G. Callejo and X. Gasull for help with DRG neuron cultures. We are grateful to D. Soto and A. Llobet for helpful discussions. MS was performed at the IRB Barcelona Mass Spectrometry Core Facility, which actively participates in the BMBS European COST Action BM 1403 and is a member of Proteored, PRB2-ISCIII, supported by grant PRB2 (IPT13/0001-ISCIII-SGEFI / FEDER). We want to thank M. Vilaseca and M. Vilanova for technical support with MS. We acknowledge financial support from the RecerCaixa foundation (2010ACUP00378); the Marató de TV3 Foundation (grants 110231 and 111531); the Human Brain Project (HBP SGA 1), the Catalan government (2012FI_B 01122, 2014SGR-1251, 2014SGR-00109 and 2009SGR-1072); the Spanish Government (SAF2012-36375, CTQ2013-43892R and CTQ2014-57020-R); the Ramón Areces foundation and the ERANET Neuron LIGHTPAIN and SynBio MODULIGHTOR projects.Peer reviewe

Optical modulation of neurotransmission using calcium photocurrents through the ion channel LiGluR

A wide range of light-activated molecules (photoswitches and phototriggers) have been used to the study of computational properties of an isolated neuron by acting pre and postsynaptically. However, new tools are being pursued to elicit a presynaptic calcium influx that triggers the release of neurotransmitters, most of them based in calcium-permeable Channelrhodopsin-2 mutants. Here we describe a method to control exocytosis of synaptic vesicles through the use of a light-gated glutamate receptor (LiGluR), which has recently been demonstrated that supports secretion by means of calcium influx in chromaffin cells. Expression of LiGluR in hippocampal neurons enables reversible control of neurotransmission with light, and allows modulating the firing rate of the postsynaptic neuron with the wavelength of illumination. This method may be useful for the determination of the complex transfer function of individual synapses

Optical modulation of neurotransmission using calcium photocurrents through the ion channel LiGluR

A wide range of light-activated molecules (photoswitches and phototriggers) have been used to the study of computational properties of an isolated neuron by acting pre and postsynaptically. However, new tools are being pursued to elicit a presynaptic calcium influx that triggers the release of neurotransmitters, most of them based in calcium-permeable Channelrhodopsin-2 mutants. Here we describe a method to control exocytosis of synaptic vesicles through the use of a light-gated glutamate receptor (LiGluR), which has recently been demonstrated that supports secretion by means of calcium influx in chromaffin cells. Expression of LiGluR in hippocampal neurons enables reversible control of neurotransmission with light, and allows modulating the firing rate of the postsynaptic neuron with the wavelength of illumination. This method may be useful for the determination of the complex transfer function of individual synapses

Optical modulation of neurotransmission using calcium photocurrents through the ion channel LiGluR

A wide range of light-activated molecules (photoswitches and phototriggers) have been used to the study of computational properties of an isolated neuron by acting pre and postsynaptically. However, new tools are being pursued to elicit a presynaptic calcium influx that triggers the release of neurotransmitters, most of them based in calcium-permeable Channelrhodopsin-2 mutants. Here we describe a method to control exocytosis of synaptic vesicles through the use of a light-gated glutamate receptor (LiGluR), which has recently been demonstrated that supports secretion by means of calcium influx in chromaffin cells. Expression of LiGluR in hippocampal neurons enables reversible control of neurotransmission with light, and allows modulating the firing rate of the postsynaptic neuron with the wavelength of illumination. This method may be useful for the determination of the complex transfer function of individual synapses

Neuronal photoactivation through second-harmonic near-infrared absorption by gold nanoparticles

Zapping neurons to life with infrared light Neuronal impulses can be generated by aiming a near-infrared laser beam at gold nanoparticles precisely tethered to brain cells. Wieteke de Boer and Jan Hirtz of the NeuroTechnology Center at Columbia University, USA, and colleagues developed the technique, which could provide a nontoxic, nongenetic alternative to commonly used optical methods for activating brain cells. They tested it in live mouse brain tissue, and also used it to induce movement in a tiny freshwater animal called Hydra vulgaris. The authors demonstrate the potential for targeted stimulation of neurons through the nonlinear absorption of light by nanoparticles. By using low-power short-pulsed near-infrared excitation, the photodamage of the tissue is minimal. The approach shows promise for application in biological systems and for future treatments of neurological and mental disorders

CACNA1A mutations causing early onset ataxia: profiling clinical, dysmorphic and structural-functional findings

The CACNA1A gene encodes the pore-forming α1A subunit of the voltage-gated CaV2.1 Ca2+ channel, essential in neurotransmission, especially in Purkinje cells. Mutations in CACNA1A result in great clinical heterogeneity with progressive symptoms, paroxysmal events or both. During infancy, clinical and neuroimaging findings may be unspecific, and no dysmorphic features have been reported. We present the clinical, radiological and evolutionary features of three patients with congenital ataxia, one of them carrying a new variant. We report the structural localization of variants and their expected functional consequences. There was an improvement in cerebellar syndrome over time despite a cerebellar atrophy progression, inconsistent response to acetazolamide and positive response to methylphenidate. The patients shared distinctive facial gestalt: oval face, prominent forehead, hypertelorism, downslanting palpebral fissures and narrow nasal bridge. The two α1A affected residues are fully conserved throughout evolution and among the whole human CaV channel family. They contribute to the channel pore and the voltage sensor segment. According to structural data analysis and available functional characterization, they are expected to exert gain- (F1394L) and loss-of-function (R1664Q/R1669Q) effect, respectively. Among the CACNA1A-related phenotypes, our results suggest that non-progressive congenital ataxia is associated with developmental delay and dysmorphic features, constituting a recognizable syndromic neurodevelopmental disorder.This work was funded by the Spanish Ministry of Health, Consumer Affairs and Social Welfare, the Spanish Ministry of Science and Innovation, the State Research Agency (AEI, Agencia Estatal de Investigación), and FEDER Funds (Fondo Europeo de Desarrollo Regional): Grants RTI2018-094809-B-I00 to J.M.F.F. and CEX2018-000792-M through the “María de Maeztu” Programme for Units of Excellence in R&D to “Departament de Ciències Experimentals i de la Salut”. M.S. is supported by the Generalitat de Catalunya (PERIS SLT008/18/00194) and National Grant PI17/00101 from the National R&D&I Plan, cofinanced by the Instituto de Salud Carlos III (Subdirectorate-General for Evaluation and Promotion of Health Research) and FEDER (European Regional Development Fund)

Adaptive selection drives TRPP3 loss-of-function in an Ethiopian population

TRPP3 (also called PKD2L1) is a nonselective, cation-permeable channel activated by multiple stimuli, including extracellular pH changes. TRPP3 had been considered a candidate for sour sensor in humans, due to its high expression in a subset of tongue receptor cells detecting sour, along with its membership to the TRP channel family known to function as sensory receptors. Here, we describe the functional consequences of two non-synonymous genetic variants (R278Q and R378W) found to be under strong positive selection in an Ethiopian population, the Gumuz. Electrophysiological studies and 3D modelling reveal TRPP3 loss-of-functions produced by both substitutions. R278Q impairs TRPP3 activation after alkalinisation by mislocation of H+ binding residues at the extracellular polycystin mucolipin domain. R378W dramatically reduces channel activity by altering conformation of the voltage sensor domain and hampering channel transition from closed to open state. Sour sensitivity tests in R278Q/R378W carriers argue against both any involvement of TRPP3 in sour detection and the role of such physiological process in the reported evolutionary positive selection past event.This work was funded by the Spanish Ministry of Science and Innovation, the State Research Agency (AEI, Agencia Estatal de Investigación) and FEDER Funds (Fondo Europeo de Desarrollo Regional): Grants BFU2016-77961-P to J.B. and E.B., RTI2018-094809-B-I00 to J.M.F.F., PID2019-110933GB-I00/AEI/10.13039/501100011033 to E.B. and J.B., and CEX2018-000792-M through the “María de Maeztu” Programme for Units of Excellence in R&D to “Departament de Ciències Experimentals i de la Salut”. With the support of Secretaria d’Universitats i Recerca del Departament d’Economia i Coneixement de la Generalitat de Catalunya (GRC 2017 SGR 702). S.W. has had a F.P.I. grant (BES-2014-068994) and M.I.-S. a “Juan de la Cierva-Incorporación” Fellowship funded by the Spanish Ministry of Science and Innovation

Two-photon neuronal and astrocytic stimulation with azobenzene-based photoswitches

This is an open access article published under an ACS AuthorChoice License. See Standard ACS AuthorChoice/Editors' Choice Usage Agreement - https://pubs.acs.org/page/policy/authorchoice_termsofuse.htmlSynthetic photochromic compounds can be designed to control a variety of proteins and their biochemical functions in living cells, but the high spatiotemporal precision and tissue penetration of two-photon stimulation have never been investigated in these molecules. Here we demonstrate two-photon excitation of azobenzene-based protein switches and versatile strategies to enhance their photochemical responses. This enables new applications to control the activation of neurons and astrocytes with cellular and subcellular resolution