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In the past decade, the optogenetic toolbox for the manipulation of ion currents and cNMP levels in Caenorhabditis elegans (C. elegans) expanded. However, the implemented tools for cAMP generation were soluble enzymes (euPAC, bPAC, IlaC22 k27 and PaaC) and thus they do not precisely mimic physiological cAMP signalling occurring in microdomains in close proximity to the plasma membrane. Here, cAMP is predominantly generated by membrane-bound adenylyl cyclases, that are located in microdomains together with G protein-coupled receptors (GPCRs), protein kinase A (PKA) and their targets, enabling spatially and temporal regulation of cAMP signalling. For this reason, one aim of this study was to develop and implement membrane bound photoactivatable adenylyl cyclases for the manipulation of cAMP mediated signalling in close proximity to the plasma membrane. For this purpose, the guanylyl cyclase domains of the Blastocladiella and Catenaria Cyclase Opsins (CyclOps) were mutated to adenylyl cyclases either by introducing the mutations E497K and C566D (abbreviated as (A-2x)) or by the mutations E497K, H564D, and C566T (abbreviated as (A-3x)).
To determine the nucleotide specificity switch from GTP to ATP and the extent of light-dependent cAMP generation, the engineered enzymes were expressed in body wall muscle cells of C. elegans and in vitro cNMP measurements using C. elegans extracts were performed. Here, the highest levels of light induced cAMP generation during sustained stimulation (0.5 mW/mm2; 470 nm, 15 min) were detected for the variants BeCyclOp(A-2x), YFP-BeCyclOp(A-2x), and YFP-CaCyclOp(A-2x) (39, 57, 40 nM, respectively), though they did not reach the extent produced by the soluble bPAC (142 nM). In contrast, low magnitudes of generated cAMP were measured for the versions BeCyclOp(A-3x) and CaCyclOp(A-2x) (8 and 7 nM, respectively). Importantly, no obvious residual cGMP and basal activity was ascertained for any of the engineered enzymes.
To assess their potential to trigger and modulate cAMP mediated cholinergic neurotransmission, and to evaluate the influence of cytosolic and membrane proximal optogenetic cAMP generation, the enzymes were expressed in cholinergic motor neurons and compared to the implemented soluble bPAC via locomotion behaviour analysis on solid and in liquid media. Photoactivation of BeCyclOp(A-2x), YFP-BeCyclOp(A-2x), and YFP-CaCyclOp(A-2x) caused similarly enhanced or even more potent behavioural changes (swimming and crawling) as bPAC, whereas a more rapidly decaying response was observed for the bPAC evoked effects. Moreover, an increased diversity of the behavioural output was detected for cytosolic cAMP production by bPAC, i.e. increased bending angles and a decreased body length.
Confocal fluorescence microscopy was performed to examine the expression levels of YFP-tagged enzymes in cholinergic neurons, whereas both YFP-CyclOp(A-2x)s were expressed at similar levels, but 1.4-fold lower relative to the soluble bPAC-YFP. To compare the amount of light-dependent cAMP generation bPAC and BeCyclOp(A-2x) at light conditions that match the conditions of the behavioural experiments (30 s), cAMP measurements using C. elegans extracts were performed, whereas BeCyclOp(A-2x) depicted a 4-fold lower amount of optogenetic cAMP production than the soluble bPAC.
In sum, local (membrane proximal) cAMP generation by the membrane-bound photoactivatable adenylyl cyclases may more specifically activate cAMP dependent neurotransmission of cholinergic motor neurons than cytosolic cAMP generation, i.e. an increased mobilization and priming/docking of synaptic vesicles and an increased filling of the synaptic vesicles with the neurotransmitter acetylcholine and thus an increase in locomotion behaviour.
The optogenetic toolbox for the manipulation of cGMP mediated signalling in C. elegans consisted of the natural membrane-bound BeCyclOp and the artificial soluble bPGC. The latter generates cGMP with low efficiency and slow kinetics (~0.2 cGMP s-1), whereas BeCyclOp enables the production of much larger amounts of cGMP (L/D = 5000) at a high turnover rate (~17 cGMP s-1). Thus, one aim of this thesis was to implement a tool with features in between those of BeCyclOp and bPGC. Several orthologous CyclOps were assessed by Gao et al., 2015 for light-regulated cGMP production by in vitro assays based on the measurement of the cNMP content from CyclOp containing oocyte membranes. Here, CaCyclOp showed the highest ratio of light versus dark activity (L/D = 230) after BeCyclOp, and thus was selected for characterization in C. elegans...
Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited disturbance of the heart rhythm (arrhythmia) that is induced by stress or that occurs during exercise. Most mutations that have been linked to CPVT are found in two genes, i.e., ryanodine receptor 2 (RyR2) and calsequestrin 2 (CASQ2), two proteins fundamentally involved in the regulation of intracellular Ca2+ in cardiac myocytes. We inserted six CPVT-causing mutations via clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 into unc-68 and csq-1, the Caenorhabditis elegans homologs of RyR and CASQ, respectively. We characterized those mutations via video-microscopy, electrophysiology, and calcium imaging in our previously established optogenetic arrhythmia model. In this study, we additionally enabled high(er) throughput recordings of intact animals by combining optogenetic stimulation with a microfluidic chip system. Whereas only minor/no pump deficiency of the pharynx was observed at baseline, three mutations of UNC-68 (S2378L, P2460S, Q4623R; RyR2-S2246L, -P2328S, -Q4201R) reduced the ability of the organ to follow 4 Hz optogenetic stimulation. One mutation (Q4623R) was accompanied by a strong reduction of maximal pump rate. In addition, S2378L and Q4623R evoked an altered calcium handling during optogenetic stimulation. The 1,4-benzothiazepine S107, which is suggested to stabilize RyR2 channels by enhancing the binding of calstabin2, reversed the reduction of pumping ability in a mutation-specific fashion. However, this depends on the presence of FKB-2, a C. elegans calstabin2 homolog, indicating the involvement of calstabin2 in the disease-causing mechanisms of the respective mutations. In conclusion, we showed for three CPVT-like mutations in C. elegans RyR a reduced pumping ability upon light stimulation, i.e., an arrhythmia-like phenotype, that can be reversed in two cases by the benzothiazepine S107 and that depends on stabilization via FKB-2. The genetically amenable nematode in combination with optogenetics and high(er) throughput recordings is a promising straightforward system for the investigation of RyR mutations and the selection of mutation-specific drugs.
The new class of microbial rhodopsins, called xenorhodopsins (XeRs),[1] extends the versatility of this family by inward H+ pumps.[2–4] These pumps are an alternative optogenetic tool to the light-gated ion channels (e.g. ChR1,2), because the activation of electrically excitable cells by XeRs is independent from the surrounding physiological conditions. In this work we functionally and spectroscopically characterized XeR from Nanosalina (NsXeR).[1] The photodynamic behavior of NsXeR was investigated on the ps to s time scale elucidating the formation of the J and K and a previously unknown long-lived intermediate. The pH dependent kinetics reveal that alkalization of the surrounding medium accelerates the photocycle and the pump turnover. In patch-clamp experiments the blue-light illumination of NsXeR in the M state shows a potential-dependent vectoriality of the photocurrent transients, suggesting a variable accessibility of reprotonation of the retinal Schiff base. Insights on the kinetically independent switching mechanism could furthermore be obtained by mutational studies on the putative intracellular H+ acceptor D220.
Background and Purpose: The cyclic nucleotides cAMP and cGMP are ubiquitous second messengers regulating numerous biological processes. Malfunctional cNMP signalling is linked to diseases and thus is an important target in pharmaceutical research. The existing optogenetic toolbox in Caenorhabditis elegans is restricted to soluble adenylyl cyclases, the membrane-bound Blastocladiella emersonii CyclOp and hyperpolarizing rhodopsins; yet missing are membrane-bound photoactivatable adenylyl cyclases and hyperpolarizers based on K+ currents.
Experimental Approach: For the characterization of photoactivatable nucleotidyl cyclases, we expressed the proteins alone or in combination with cyclic nucleotide-gated channels in muscle cells and cholinergic motor neurons. To investigate the extent of optogenetic cNMP production and the ability of the systems to depolarize or hyperpolarize cells, we performed behavioural analyses, measured cNMP content in vitro, and compared in vivo expression levels.
Key Results: We implemented Catenaria CyclOp as a new tool for cGMP production, allowing fine-control of cGMP levels. We established photoactivatable membrane-bound adenylyl cyclases, based on mutated versions (“A-2x”) of Blastocladiella and Catenaria (“Be,” “Ca”) CyclOp, as N-terminal YFP fusions, enabling more efficient and specific cAMP signalling compared to soluble bPAC, despite lower overall cAMP production. For hyperpolarization of excitable cells by two-component optogenetics, we introduced the cAMP-gated K+-channel SthK from Spirochaeta thermophila and combined it with bPAC, BeCyclOp(A-2x), or YFP-BeCyclOp(A-2x). As an alternative, we implemented the B. emersonii cGMP-gated K+-channel BeCNG1 together with BeCyclOp.
Conclusion and Implications: We established a comprehensive suite of optogenetic tools for cNMP manipulation, applicable in many cell types, including sensory neurons, and for potent hyperpolarization.
Optogenetic stimulation of inhibitory interneurons has become a commonly used strategy for silencing neuronal activity. This is typically achieved using transgenic mice expressing excitatory opsins in inhibitory interneurons throughout the brain, raising the question of how spatially extensive the resulting inhibition is. Here, we characterize neuronal silencing in VGAT-ChR2 mice, which express channelrhodopsin-2 in inhibitory interneurons, as a function of light intensity and distance from the light source in several cortical and subcortical regions. We show that light stimulation, even at relatively low intensities, causes inhibition not only in brain regions targeted for silencing but also in their subjacent areas. In contrast, virus-mediated expression of an inhibitory opsin enables robust silencing that is restricted to the region of opsin expression. Our results reveal important constraints on using inhibitory interneuron activation to silence neuronal activity and emphasize the necessity of carefully controlling light stimulation parameters when using this silencing strategy.