Optogenetic induction of chronic glucocorticoid exposure in early-life leads to blunted stress-response in larval zebrafish

Early life stress (ELS) exposure alters stress susceptibility in later life and affects vulnerability to stress-related disorders, but how ELS changes the long-lasting responsiveness of the stress system is not well understood. Zebrafish provides an opportunity to study conserved mechanisms underlying the development and function of the stress response that is regulated largely by the neuroendocrine hypothalamus-pituitary-adrenal/interrenal (HPA/I) axis, with glucocorticoids (GC) as the final effector. In this study, we established a method to chronically elevate endogenous GC levels during early life in larval zebrafish. To this end, we employed an optogenetic actuator, beggiatoa photoactivated adenylyl cyclase, specifically expressed in the interrenal cells of zebrafish and demonstrate that its chronic activation leads to hypercortisolaemia and dampens the acute-stress evoked cortisol levels, across a variety of stressor modalities during early life. This blunting of stress-response was conserved in ontogeny at a later developmental stage. Furthermore, we observe a strong reduction of proopiomelanocortin (pomc)-expression in the pituitary as well as upregulation of fkbp5 gene expression. Going forward, we propose that this model can be leveraged to tease apart the mechanisms underlying developmental programming of the HPA/I axis by early-life GC exposure and its implications for vulnerability and resilience to stress in adulthood

Optogenetic tools for manipulation of cyclic nucleotides functionally coupled to cyclic nucleotide‐gated channels

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.Deutsche Forschungsgemeinschaft (DFG) http://dx.doi.org/10.13039/501100001659Peer Reviewe

Neurofilament Heavy Polypeptide Regulates the Akt-β-Catenin Pathway in Human Esophageal Squamous Cell Carcinoma

Aerobic glycolysis and mitochondrial dysfunction are common features of aggressive cancer growth. We observed promoter methylation and loss of expression in neurofilament heavy polypeptide (NEFH) in a significant proportion of primary esophageal squamous cell carcinoma (ESCC) samples that were of a high tumor grade and advanced stage. RNA interference-mediated knockdown of NEFH accelerated ESCC cell growth in culture and increased tumorigenicity in vivo, whereas forced expression of NEFH significantly inhibited cell growth and colony formation. Loss of NEFH caused up-regulation of pyruvate kinase-M2 type and down-regulation of pyruvate dehydrogenase, via activation of the Akt/β-catenin pathway, resulting in enhanced aerobic glycolysis and mitochondrial dysfunction. The acceleration of glycolysis and mitochondrial dysfunction in NEFH-knockdown cells was suppressed in the absence of β-catenin expression, and was decreased by the treatment of 2-Deoxyglucose, a glycolytic inhibitor, or API-2, an Akt inhibitor. Loss of NEFH activates the Akt/β-catenin pathway and increases glycolysis and mitochondrial dysfunction. Cancer cells with methylated NEFH can be targeted for destruction with specific inhibitors of deregulated downstream pathways

Using a Robust and Sensitive GFP-Based cGMP Sensor for Real-Time Imaging in Intact Caenorhabditis elegans.

cGMP plays a role in sensory signaling and plasticity by regulating ion channels, phosphodiesterases, and kinases. Studies that primarily used genetic and biochemical tools suggest that cGMP is spatiotemporally regulated in multiple sensory modalities. FRET- and GFP-based cGMP sensors were developed to visualize cGMP in primary cell culture and Caenorhabditis elegans to corroborate these findings. While a FRET-based sensor has been used in an intact animal to visualize cGMP, the requirement of a multiple emission system limits its ability to be used on its own as well as with other fluorophores. Here, we demonstrate that a C. elegans codon-optimized version of the cpEGFP-based cGMP sensor FlincG3 can be used to visualize rapidly changing cGMP levels in living, behaving C. elegans We coexpressed FlincG3 with the blue-light-activated guanylyl cyclases BeCyclOp and bPGC in body wall muscles, and found that the rate of change in FlincG3 fluorescence correlated with the rate of cGMP production by each cyclase. Furthermore, we show that FlincG3 responds to cultivation temperature, NaCl concentration changes, and sodium dodecyl sulfate in the sensory neurons AFD, ASEL/R, and PHB, respectively. Intriguingly, FlincG3 fluorescence in ASEL and ASER decreased in response to a NaCl concentration upstep and downstep, respectively, which is opposite in sign to the coexpressed calcium sensor jRGECO1a and previously published calcium recordings. These results illustrate that FlincG3 can be used to report rapidly changing cGMP levels in an intact animal, and that the reporter can potentially reveal unexpected spatiotemporal landscapes of cGMP in response to stimuli

ssDNA-binding protein 2 is frequently hypermethylated and suppresses cell growth in human prostate cancer

Purpose: Prostate cancer is a major cause of cancer death among men and the development of new biomarkers is important to augment current detection approaches. Experimental Design: We identified hypermethylation of the ssDNA-binding protein 2 (SSBP2) promoter as a potential DNA marker for human prostate cancer based on previous bioinformatics results and pharmacologic unmasking microarray. We then did quantitative methylation-specific PCR in primary prostate cancer tissues to confirm hypermethylation of the SSBP2 promoter, and analyzed its correlation with clinicopathologic data. We further examined SSBP2 expression in primary prostate cancer and studied its role in cell growth. Results: Quantitative methylation-specific PCR results showed that the SSBP2 promoter was hypermethylated in 54 of 88 (61.4%) primary prostate cancers versus 0 of 23 (0%) in benign prostatic hyperplasia using a cutoff value of 120. Furthermore, we found that expression of SSBP2 was down-regulated in primary prostate cancers and cancer cell lines. Hypermethylation of the SSBP2 promoter and its expression were closely associated with higher stages of prostate cancer. Reactivation of SSBP2 expression by the demethylating agent 5-aza-2'-deoxycytidine in prostate cancer cell lines confirmed epigenetic inactivation as one major mechanism of SSBP2 regulation. Moreover, forced expression of SSBP2 inhibited prostate cancer cell proliferation in the colony formation assay and caused cell cycle arrest. Conclusion: SSBP2 inhibits prostate cancer cell proliferation and seems to represent a novel prostate cancer - specific DNA marker, especially in high stages of human prostate cancer

Photoswitchable diacylglycerols enable optical control of protein kinase C.

Increased levels of the second messenger lipid diacylglycerol (DAG) induce downstream signaling events including the translocation of C1-domain-containing proteins toward the plasma membrane. Here, we introduce three light-sensitive DAGs, termed PhoDAGs, which feature a photoswitchable acyl chain. The PhoDAGs are inactive in the dark and promote the translocation of proteins that feature C1 domains toward the plasma membrane upon a flash of UV-A light. This effect is quickly reversed after the termination of photostimulation or by irradiation with blue light, permitting the generation of oscillation patterns. Both protein kinase C and Munc13 can thus be put under optical control. PhoDAGs control vesicle release in excitable cells, such as mouse pancreatic islets and hippocampal neurons, and modulate synaptic transmission in Caenorhabditis elegans. As such, the PhoDAGs afford an unprecedented degree of spatiotemporal control and are broadly applicable tools to study DAG signaling

Development and implementation of novel optogenetic tools in the nematode Caenorhabditis elegans

Optogenetics, though still only a decade old field, has revolutionized research in neurobiology. It comprises of methods that allow control of neural activity by light in a minimally-invasive, spatio-temporally precise and genetically targeted manner. The optogenetic actuators or the genetically encoded light sensitive elements mediate light driven manipulation of membrane potential, intracellular signalling, neuronal network activity and behaviour (Fenno et al. 2011; Dugué et al. 2012). These techniques have been particularly useful for dissecting neural circuits and behaviour in the transparent and genetically amenable nematode model system Caenorhabditis elegans (Husson et al. 2013; Fang-yen et al. 2015). In fact, C. elegans was the first living organism in which microbial rhodopsin based optogenetic tools (Channelrhodopsin-2 or ChR2, and Halorhodopsin or NpHR) were successfully implemented and bimodal 'remote' control of behaviour was achieved (Nagel et al. 2005; Zhang et al. 2007). Since then it has been a prominent model for the development and application of novel optogenetic tools and techniques, especially in the nervous system which comprises of 302 neurons and is organised in a hierarchical organization. The environmental stimuli are sensed by the sensory neurons, leading to the processing of information by the downstream interneurons, that relay to motor neurons which in-turn synapse onto muscles that drive the movement-based responses. The microbial rhodopsins like ChR2 and NpHR mediate light driven depolarization and hyperpolarization, respectively and thereby activate or inhibit neural activity. However, they do not allow local control of membrane potential as they are expressed all over the plasma membrane of the cell rather than being restricted to specific domains, for example synaptic sites. Moreover, they completely over-ride the intrinsic activity of the cell, completely bypassing the signal transduction processes inside the cell. Thus, in order to study intracellular signalling and to answer questions pertaining to the endogenous role of receptors and channels in an in-vivo context, the optogenetic tool-kit needs to be expanded. This thesis aimed at developing and implementing novel optogenetic tools in C. elegans that allow for sub-cellular signalling control as well as endogenous receptor control. These are: two light activated guanylyl cyclases (bPGC and BeCyclOp) to modify cyclic guanosine monophosphate (cGMP) mediated signalling in the sensory neurons, as well as attempts towards rendering endogenous C. elegans receptors - glutamate receptor (GLR-3/-6), acetylcholine receptor (ACR-16), glutamate gated chloride channel (GLC-1) light switchable and to understand their biological function in-vivo. Organisms respond to sensory cues by activation of a primary receptor followed by relay of information downstream to effector targets by secondary signalling molecules. cGMP is a widely used 2nd messenger in cellular signaling, acting via protein kinase G or cyclic nucleotide gated (CNG) channels. In sensory neurons, cGMP allows for signal modulation and amplification, before depolarization. Chemo-, thermo-, and oxygen-sensation in C. elegans involve sensory neurons that use cGMP as the main 2nd messenger. For example, ASJ is the pheromone sensing neuron regulating larval development, AWC is the chemosensory neuron responding to volatile odours and BAG senses oxygen and carbon dioxide in the environment. In these neurons, cGMP acts downstream of the GPCRs and functions by activating cationic TAX-2/-4 CNG channels, thereby depolarising the sensory neuron. Manipulating cGMP levels is required to access signalling between sensation and sensory neuron depolarization, thereby provide insights into signal encoding. We achieve this by implementing two photo-activatable guanylyl cyclases - 1) a mutated version of Beggiatoa sp. bacterial light-activated adenylyl cyclase, with specificity for GTP (Ryu et al. 2010), termed BlgC or bPGC (Beggiatoa photoactivated guanylyl cyclase) and 2) guanylyl cyclase rhodopsin (Avelar et al. 2014) from Blastocladiella emersonii (BeCyclOp). bPGC is a BLUF (blue light sensing using flavin) domain containing cyclase which uses FAD as the co-factor and catalyses the synthesis of cGMP from GTP upon activation by blue light. Prior to implementation in sensory neurons, a simpler heterologous system with co-expression of the TAX-2/-4 CNG channel in C. elegans body wall muscle (BWM) was used. The cGMP generated by the light activated cyclases activates the CNG channel leading to the muscle depolarization, thereby causing changes in body length which can be easily scored.Obwohl die Optogenetik ein nur zehn Jahre altes Forschungsgebiet ist, hat sie die Forschung in der Neurobiologie revolutioniert. Sie umfasst Methoden, die die Kontrolle neuronaler Aktivität durch Licht in einer minimal-invasiven, räumlich und zeitlich präzisen, und genetisch gezielten Weise ermöglichen. Die optogenetischen Aktoren, auch als genetisch codierte, lichtempfindliche Elemente zu beschreiben, ermöglichen, durch Licht angetrieben, die Manipulation von Membranpotentialen, von intrazellulären Signalwegen, sowie von neuronalen Netzwerkaktivitäten und Verhalten (Fenno et al. 2011; Dugué et al. 2012). Diese Techniken haben sich als besonders nützlich für die Sektion von neuronalen Schaltkreisen und des Verhaltens in der Nematode Caenorhabditis elegans erwiesen; einem transparenten und genetisch zugänglichen Modellsystem (Husson et al. 2013; Fang-yen et al. 2015). Tatsächlich ist C. elegans der erste Organismus, in dem auf mikrobiellem Rhodopsin basierende optogenetische Werkzeuge (Channelrhodopsin-2 oder ChR2 und Halorhodopsin oder NpHR) erfolgreich implementiert wurden und so auch eine bimodale ferngesteuerter Kontrolle des Verhaltens erreicht wurde (Nagel et al. 2005; Zhang et al. 2007). Seitdem ist der Nematode ein hervorragendes Modell für die Entwicklung und Anwendung von neuartigen optogenetischen Werkzeugen und Techniken, vor allem in seinem Nervensystem, welches aus 302 Neuronen besteht und in einer hierarchischen Weise organisiert ist. Stimuli aus der Umgebung werden von den sensorischen Neuronen erkannt, führen zu einer Weiterleitung der Informationen durch die stromabwärts liegenden Interneuronen zu den Motorneuronen, welche synaptisch mit den Muskeln verbunden sind und dort eine bewegungsbasierte Reaktion erwirken. Mikrobielle Rhodopsine wie ChR2 und NpHR vermitteln jeweils Licht angetriebene Depolarisation und Hyperpolarisation, wodurch sie neuronale Aktivität induzieren oder unterdrücken. Sie erlauben jedoch keine lokale Kontrolle des Membranpotentials, da sie überall in der Plasmamembran der Zellen exprimieren und deswegen nicht auf bestimmte Domänen beschränkt sind, wie beispielsweise in den Synapsen. Zudem überschreiten sie die intrinsische Aktivität der Zelle und umgehen so auch die Signaltransduktionsprozesse innerhalb der Zelle. Somit muss, um die intrazellulären Signalwege zu studieren und um die Fragen über die endogene Rolle von Rezeptoren und Kanälen in einem in vivo Kontext zu beantworten, die Sammlung optogenetischer Werkzeuge erweitert werden. Das Ziel dieser Arbeit ist die Entwicklung und Umsetzung neuartiger optogenetischer Werkzeuge in C. elegans, welche die Kontrolle der subzellulären Signalsteuerung sowie die von endogenen Rezeptoren ermöglichen. Diese sind: zwei Licht-aktivierte Guanylylzyklasen (bPGC und BeCyclOp), um die durch zyklisches Guanosinmonophosphat (cGMP) vermittelten Signalwege in den sensorischen Neuronen zu modifizieren, sowie Versuche die endogenen C. elegans Rezeptoren - Glutamatrezeptor (GLR-3/-6), Acetylcholin - Rezeptor (ACR-16) und Glutamat gesteuerter Chlorid - Kanal (GLC-1) lichtschaltbar zu machen und ihre biologische Funktion in vivo zu verstehen. Organismen reagieren auf sensorische Signale durch die Aktivierung eines primären Rezeptors, gefolgt von der Übertragung der Information stromabwärts durch sekundäre Signalisierungsmoleküle zum Wirkungsort. cGMP ist ein weit verbreiteter sekundärer Botenstoff in der zellulären Signaltransduktion, der über Proteinkinase G oder zyklische Nukleotid gesteuerte (CNG) Kanäle wirkt. In sensorischen Neuronen ermöglicht cGMP vor einer Depolarisation eine Modulation und Verstärkung des Signals. Die Chemo-, Thermo- und Sauerstoff-Erkennung in C. elegans involviert die sensorischen Neuronen, welche hauptsächlich cGMP als sekundären Botenstoff benutzen. Zum Beispiel reguliert das Pheromon erkennende ASJ Neuron die Larvenentwicklung, reagiert das chemosensorische Neuron AWC auf flüchtige Gerüche und BAG detektiert Sauerstoff und Kohlendioxid in der Umgebung. In diesen Neuronen wirkt cGMP stromabwärts der GPCRs und aktiviert die kationischen TAX-2/-4 CNG - Kanäle, wodurch die sensorischen Neuronen depolarisiert werden. Die Manipulation der cGMP Niveaus ist erforderlich um auf die Signale zwischen Reizerkennung und Depolarisation von sensorischen Neuronen zuzugreifen und Einblick in die Signalkodierung zu erlangen. Dies erreichen wir durch das Implementieren zweier photoaktivierbarer Guanylylzyclasen - 1) einer mutierten Version der Beggiatoa sp bakteriellen, lichtaktivierten Adenylatzyklase, mit Spezifität für GTP (Ryu et al. 2010), die BlgC oder bPGC genannt wird (Beggiatoa photoaktivierte Guanylylzyklase) und 2) einem Guanylatzyklase-Rhodopsin (Avelar et al. 2014) von Blastocladiella emersonii (BeCyclOp)

Microbiota-brain interactions: Moving toward mechanisms in model organisms

Changes in the microbiota are associated with alterations in nervous system structure-function and behavior and have been implicated in the etiology of neuropsychiatric and neurodegenerative disorders. Most of these studies have centered on mammalian models due to their phylogenetic proximity to humans. Indeed, the germ-free mouse has been a particularly useful model organism for investigating microbiota-brain interactions. However, microbiota-brain axis research on simpler genetic model organisms with a vast and diverse scientific toolkit (zebrafish, Drosophila melanogaster, and Caenorhabditis elegans) is now also coming of age. In this review, we summarize the current state of microbiota-brain axis research in rodents and humans, and then we elaborate and discuss recent research on the neurobiological and behavioral effects of the microbiota in the model systems of fish, flies, and worms. We propose that a cross-species, holistic and mechanistic approach to unravel the microbiota-brain communication is an essential step toward rational microbiota-based therapeutics to combat brain disorders