Multi-faceted regulation of CREB family transcription factors.

cAMP response element-binding protein (CREB) is a ubiquitously expressed nuclear transcription factor, which can be constitutively activated regardless of external stimuli or be inducibly activated by external factors such as stressors, hormones, neurotransmitters, and growth factors. However, CREB controls diverse biological processes including cell growth, differentiation, proliferation, survival, apoptosis in a cell-type-specific manner. The diverse functions of CREB appear to be due to CREB-mediated differential gene expression that depends on cAMP response elements and multi-faceted regulation of CREB activity. Indeed, the transcriptional activity of CREB is controlled at several levels including alternative splicing, post-translational modification, dimerization, specific transcriptional co-activators, non-coding small RNAs, and epigenetic regulation. In this review, we present versatile regulatory modes of CREB family transcription factors and discuss their functional consequences.

Function and regulation of nitric oxide signaling in Drosophila.

Nitric oxide (NO) serves as an evolutionarily conserved signaling molecule that plays an important role in a wide variety of cellular processes. Extensive studies in Drosophila melanogaster have revealed that NO signaling is required for development, physiology, and stress responses in many different types of cells. In neuronal cells, multiple NO signaling pathways appear to operate in different combinations to regulate learning and memory formation, synaptic transmission, selective synaptic connections, axon degeneration, and axon regrowth. During organ development, elevated NO signaling suppresses cell cycle progression, whereas downregulated NO leads to an increase in larval body size via modulation of hormone signaling. The most striking feature of the Drosophila NO synthase is that various stressors, such as neuropeptides, aberrant proteins, hypoxia, bacterial infection, and mechanical injury, can activate Drosophila NO synthase, initially regulating cellular physiology to enable cells to survive. However, under severe stress or pathophysiological conditions, high levels of NO promote regulated cell death and the development of neurodegenerative diseases. In this review, I highlight and discuss the current understanding of molecular mechanisms by which NO signaling regulates distinct cellular functions and behaviors.

Facile Fabrication of TiO2 Quantum Dots-Anchored g-C3N4 Nanosheets as 0D/2D Heterojunction Nanocomposite for Accelerating Solar-Driven Photocatalysis.

TiO2 semiconductors exhibit a low catalytic activity level under visible light because of their large band gap and fast recombination of electron-hole pairs. This paper reports the simple fabrication of a 0D/2D heterojunction photocatalyst by anchoring TiO2 quantum dots (QDs) on graphite-like C3N4 (g-C3N4) nanosheets (NSs); the photocatalyst is denoted as TiO2 QDs@g-C3N4. The nanocomposite was characterized via analytical instruments, such as powder X-ray diffraction, X-ray photoelectron spectroscopy, scanning electron microscopy, transmission electron microscopy, t orange (MO) under solar light were compared. The TiO2 QDs@g-C3N4 photocatalyst exhibited 95.57% MO degradation efficiency and ~3.3-fold and 5.7-fold higher activity level than those of TiO2 QDs and g-C3N4 NSs, respectively. Zero-dimensional/two-dimensional heterojunction formation with a staggered electronic structure leads to the efficient separation of photogenerated charge carriers via a Z-scheme pathway, which significantly accelerates photocatalysis under solar light. This study provides a facile synthetic method for the rational design of 0D/2D heterojunction nanocomposites with enhanced solar-driven catalytic activity.

The Pleiotropic Face of CREB Family Transcription Factors.

cAMP responsive element-binding protein (CREB) is one of the most intensively studied phosphorylation-dependent transcription factors that provide evolutionarily conserved mechanisms of differential gene expression in vertebrates and invertebrates. Many cellular protein kinases that function downstream of distinct cell surface receptors are responsible for the activation of CREB. Upon functional dimerization of the activated CREB to cis-acting cAMP responsive elements within the promoters of target genes, it facilitates signal-dependent gene expression. From the discovery of CREB, which is ubiquitously expressed, it has been proven to be involved in a variety of cellular processes that include cell proliferation, adaptation, survival, differentiation, and physiology, through the control of target gene expression. In this review, we highlight the essential roles of CREB proteins in the nervous system, the immune system, cancer development, hepatic physiology, and cardiovascular function and further discuss a wide range of CREB-associated diseases and molecular mechanisms underlying the pathogenesis of these diseases.

Regulation of Off-track bidirectional signaling by Semaphorin-1a and Wnt signaling in the Drosophila motor axon guidance.

Off-track receptor tyrosine kinase (OTK) has been shown to play an important role in the Drosophila motor axon pathfinding. The results of biochemical and genetic interactions previously suggested that OTK acts as a component of Semaphorin-1a/Plexin A (Sema-1a/PlexA) signaling during embryonic motor axon guidance and further showed that OTK binds to Wnt family members Wnt2 and Wnt4 and their common receptor Frizzled (Fz). However, the molecular mechanisms underlying the motor axon guidance function of OTK remain elusive. Here, we conclude that OTK mediates the forward and reverse signaling required for intersegmental nerve b (ISNb) motor axon pathfinding and we also demonstrate that the loss of two copies of Sema-1a synergistically enhances the bypass phenotype observed in otk mutants. Furthermore, the amorphic wnt2 mutation resulted in increased premature branching phenotypes, and the loss of fz function caused a frequent inability of ISNb motor axons to defasciculate at specific choice points. Consistent with a previous study, wnt4 mutant axons were often defective in recognizing target muscles. Interestingly, the bypass phenotype of otk mutants was robustly suppressed by loss of function mutations in wnt2, wnt4, or fz. In contrast, total ISNb defects of otk were increased by the loss-of-function alleles in wnt2 and wnt4, but not fz. These findings indicate that OTK may participate in the crosstalk between the Sema-1a/PlexA and Wnt signaling pathways, thereby contributing to ISNb motor axon pathfinding and target recognition.

A Report on Multi-Target Anti-Inflammatory Properties of Phytoconstituents from Monochoria hastata (Family: Pontederiaceae).

This study aims to investigate the potential analgesic properties of the crude extract of Monochoria hastata (MH) leaves using in vivo experiments and in silico analysis. The extract, in a dose-dependent manner, exhibited a moderate analgesic property (~54% pain inhibition in acetic acid-induced writhing test), which is significant (** p < 0.001) as compared to the control group. The complex inflammatory mechanism involves diverse pathways and they are inter-connected. Therefore, multiple inflammatory modulator proteins were selected as the target for in silico analysis. Computational analysis suggests that all the selected targets had different degrees of interaction with the phytochemicals from the extract. Rutin (RU), protocatechuic acid (PA), vanillic acid (VA), and ferulic acid (FA) could regulate multiple targets with a robust efficiency. None of the compounds showed selectivity to Cyclooxygenase-2 (COX-2). However, regulation of COX and lipoxygenase (LOX) cascade by PA can reduce non-steroidal analgesic drugs (NSAIDs)-related side effects, including asthma. RU showed robust regulation of cytokine-mediated pathways like RAS/MAPK and PI3K/NF-kB by inhibition of EGFR and IKBα (IKK), which may prevent multi-organ failure due to cytokine storm in several microbial infections, for example, SARS-CoV-2. Further investigation, using in vivo and in vitro experiments, can be conducted to develop multi-target anti-inflammatory drugs using the isolated compounds from the extract.

Molecular Mechanisms Underlying Motor Axon Guidance in Drosophila.

Decoding the molecular mechanisms underlying axon guidance is key to precise understanding of how complex neural circuits form during neural development. Although substantial progress has been made over the last three decades in identifying numerous axon guidance molecules and their functional roles, little is known about how these guidance molecules collaborate to steer growth cones to their correct targets. Recent studies in Drosophila point to the importance of the combinatorial action of guidance molecules, and further show that selective fasciculation and defasciculation at specific choice points serve as a fundamental strategy for motor axon guidance. Here, I discuss how attractive and repulsive guidance cues cooperate to ensure the recognition of specific choice points that are inextricably linked to selective fasciculation and defasciculation, and correct pathfinding decision-making.

Identification of cis -Regulatory Region Controlling Semaphorin-1a Expression in the Drosophila Embryonic Nervous System.

The Drosophila transmembrane semaphorin Sema-1a mediates forward and reverse signaling that plays an essential role in motor and central nervous system (CNS) axon pathfinding during embryonic neural development. Previous immunohistochemical analysis revealed that Sema-1a is expressed on most commissural and longitudinal axons in the CNS and five motor nerve branches in the peripheral nervous system (PNS). However, Sema-1a-mediated axon guidance function contributes significantly to both intersegmental nerve b (ISNb) and segmental nerve a (SNa), and slightly to ISNd and SNc, but not to ISN motor axon pathfinding. Here, we uncover three cis-regulatory elements (CREs), R34A03, R32H10, and R33F06, that robustly drove reporter expression in a large subset of neurons in the CNS. In the transgenic lines R34A03 and R32H10 reporter expression was consistently observed on both ISNb and SNa nerve branches, whereas in the line R33F06 reporter expression was irregularly detected on ISNb or SNa nerve branches in small subsets of abdominal hemisegments. Through complementation test with a Sema1a loss-of-function allele, we found that neuronal expression of Sema-1a driven by each of R34A03 and R32H10 restores robustly the CNS and PNS motor axon guidance defects observed in Sema-1a homozygous mutants. However, when wild-type Sema-1a is expressed by R33F06 in Sema-1a mutants, the Sema-1a PNS axon guidance phenotypes are partially rescued while the Sema-1a CNS axon guidance defects are completely rescued. These results suggest that in a redundant manner, the CREs, R34A03, R32H10, and R33F06 govern the Sema-1a expression required for the axon guidance function of Sema-1a during embryonic neural development.

Molecular and Cellular Basis of Neurodegeneration in Alzheimer's Disease.

The most common form of senile dementia is Alzheimer's disease (AD), which is characterized by the extracellular deposition of amyloid ß-peptide (Aß) plaques and the intracellular formation of neurofibrillary tangles (NFTs) in the cerebral cortex. Tau abnormalities are commonly observed in many neurodegenerative diseases including AD, Parkinson's disease, and Pick's disease. Interestingly, tau-mediated formation of NFTs in AD brains shows better correlation with cognitive impairment than Aß plaque accumulation; pathological tau alone is sufficient to elicit frontotemporal dementia, but it does not cause AD. A growing amount of evidence suggests that soluble Aß oligomers in concert with hyperphosphorylated tau (pTau) serve as the major pathogenic drivers of neurodegeneration in AD. Increased Aß oligomers trigger neuronal dysfunction and network alternations in learning and memory circuitry prior to clinical onset of AD, leading to cognitive decline. Furthermore, accumulated damage to mitochondria in the course of aging, which is the best-known nongenetic risk factor for AD, may collaborate with soluble Aß and pTau to induce synapse loss and cognitive impairment in AD. In this review, I summarize and discuss the current knowledge of the molecular and cellular biology of AD and also the mechanisms that underlie Aß-mediated neurodegeneration.

Varicose and cheerio collaborate with pebble to mediate semaphorin-1a reverse signaling in Drosophila.

The transmembrane semaphorin Sema-1a acts as both a ligand and a receptor to regulate axon-axon repulsion during neural development. Pebble (Pbl), a Rho guanine nucleotide exchange factor, mediates Sema-1a reverse signaling through association with the N-terminal region of the Sema-1a intracellular domain (ICD), resulting in cytoskeletal reorganization. Here, we uncover two additional Sema-1a interacting proteins, varicose (Vari) and cheerio (Cher), each with neuronal functions required for motor axon pathfinding. Vari is a member of the membrane-associated guanylate kinase (MAGUK) family of proteins, members of which can serve as scaffolds to organize signaling complexes. Cher is related to actin filament cross-linking proteins that regulate actin cytoskeleton dynamics. The PDZ domain binding motif found in the most C-terminal region of the Sema-1a ICD is necessary for interaction with Vari, but not Cher, indicative of distinct binding modalities. Pbl/Sema-1a-mediated repulsive guidance is potentiated by both vari and cher Genetic analyses further suggest that scaffolding functions of Vari and Cher play an important role in Pbl-mediated Sema-1a reverse signaling. These results define intracellular components critical for signal transduction from the Sema-1a receptor to the cytoskeleton and provide insight into mechanisms underlying semaphorin-induced localized changes in cytoskeletal organization.

Gustatory receptor 22e is essential for sensing chloroquine and strychnine in Drosophila melanogaster.

Chloroquine, an amino quinolone derivative commonly used as an anti-malarial drug, is known to impart an unpleasant taste. Little research has been done to study chloroquine taste in insects, therefore, we examined both the deterrant properties and mechanisms underlying chloroquine perception in fruit flies. We identified the antifeedant effect of chloroquine by screening 21 gustatory receptor (Grs) mutants through behavioral feeding assays and electrophysiology experiments. We discovered that two molecular sensors, GR22e and GR33a, act as chloroquine receptors, and found that chloroquine-mediated activation of GRNs occurs through S-type sensilla. At the same time, we successfully recapitulated the chloroquine receptor by expressing GR22e in ectopic gustatory receptor neurons. We also found that GR22e forms a part of the strychnine receptor. We suggest that the Drosophila strychnine receptor might have a very complex structure since five different GRs are required for strychnine-induced action potentials.

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila.

The establishment of functional neuromuscular circuits relies on precise connections between developing motor axons and target muscles. Motor neurons extend growth cones to navigate along specific pathways by responding to a large number of axon guidance cues that emanate from the surrounding extracellular environment. Growth cone target recognition also plays a critical role in neuromuscular specificity. This work presents a standard immunohistochemistry protocol to visualize motor neuron projections of late stage-16 Drosophila melanogaster embryos. This protocol includes a few key steps, including a genotyping procedure, to sort the desired mutant embryos; an immunostaining procedure, to tag embryos with fasciclin II (FasII) antibody; and a dissection procedure, to generate filleted preparations from fixed embryos. Motor axon projections and muscle patterns in the periphery are much better visualized in flat preparations of filleted embryos than in whole-mount embryos. Therefore, the filleted preparation of fixed embryos stained with FasII antibody provides a powerful tool to characterize the genes required for motor axon pathfinding and target recognition, and it can also be applied to both loss-of-function and gain-of-function genetic screens.

Dexamethasone suppresses JMJD3 gene activation via a putative negative glucocorticoid response element and maintains integrity of tight junctions in brain microvascular endothelial cells.

The blood-brain barrier (BBB) exhibits a highly selective permeability to support the homeostasis of the central nervous system (CNS). The tight junctions in the BBB microvascular endothelial cells seal the paracellular space to prevent diffusion. Thus, disruption of tight junctions results in harmful effects in CNS diseases and injuries. It has recently been demonstrated that glucocorticoids have beneficial effects on maintaining tight junctions in both in vitro cell and in vivo animal models. In the present study, we found that dexamethasone suppresses the expression of JMJD3, a histone H3K27 demethylase, via the recruitment of glucocorticoid receptor α (GRα) and nuclear receptor co-repressor (N-CoR) to the negative glucocorticoid response element (nGRE) in the upstream region of JMJD3 gene in brain microvascular endothelial cells subjected to TNFα treatment. The decreased JMJD3 gene expression resulted in the suppression of MMP-2, MMP-3, and MMP-9 gene activation. Dexamethasone also activated the expression of the claudin 5 and occludin genes. Collectively, dexamethasone attenuated the disruption of the tight junctions in the brain microvascular endothelial cells subjected to TNFα treatment. Therefore, glucocorticoids may help to preserve the integrity of the tight junctions in the BBB via transcriptional and post-translational regulation following CNS diseases and injuries.

Development of a Reporter System for In Vivo Monitoring of γ-Secretase Activity in Drosophila.

The γ-secretase complex represents an evolutionarily conserved family of transmembrane aspartyl proteases that cleave numerous type-I membrane proteins, including the ß-amyloid precursor protein (APP) and the receptor Notch. All known rare mutations in APP and the γ-secretase catalytic component, presenilin, which lead to increased amyloid ßpeptide production, are responsible for early-onset familial Alzheimer's disease. ß-amyloid protein precursor-like (APPL) is the Drosophila ortholog of human APP. Here, we created Notch- and APPL-based Drosophila reporter systems for in vivo monitoring of γ-secretase activity. Ectopic expression of the Notch- and APPL-based chimeric reporters in wings results in vein truncation phenotypes. Reporter-mediated vein truncation phenotypes are enhanced by the Notch gain-of-function allele and suppressed by RNAi-mediated knockdown of presenilin. Furthermore, we find that apoptosis partly contributes to the vein truncation phenotypes of the APPL-based reporter, but not to the vein truncation phenotypes of the Notch-based reporter. Taken together, these results suggest that both in vivo reporter systems provide a powerful genetic tool to identify genes that modulate γ-secretase activity and/or APPL metabolism.

Differential ligand regulation of PlexB signaling in motor neuron axon guidance in Drosophila.

Plexins (Plexs) are a large family of phylogenetically conserved guidance receptors that bind specifically to semaphorins (Semas), another large family of guidance molecules. In the Drosophila embryonic central nervous system (CNS), the secreted semaphorins Sema-2a and Sema-2b both act as ligands for PlexB, but mediate mutually independent and opposite functions (repulsive and attractive guidance, respectively). PlexB is also known to regulate motor axon guidance in the embryonic peripheral nervous system (PNS). However, it is unclear whether the mechanisms of ligand regulation of PlexB seen in the CNS are similar or the same as those that exist in PNS motor axon guidance. Here, we find that two distinct modes of ligand regulation underlie differential roles of PlexB in PNS motor axon pathfinding during embryonic development. Epistasis analyses in the intersegmental nerve b (ISNb) pathway suggest that PlexB serves as a receptor for both Sema-2a and Sema-2b and integrates their mutually dependent but opposite guidance functions. Furthermore, we present evidence that PlexB mediates not only Sema-2a/2b-dependent guidance functions, but also Sema-2a/2b-independent target recognition in establishing the segmental nerve a (SNa) motor axon pathway. These results demonstrate that a single guidance receptor can elicit diverse effects on the establishment of neuronal connectivity via regulation of its ligands themselves.

The axon guidance function of Rap1 small GTPase is independent of PlexA RasGAP activity in Drosophila.

Plexins (Plexs) comprise a large family of cell surface receptors for semaphorins (Semas) that function as evolutionarily conserved guidance molecules. GTPase activating protein (GAP) activity for Ras family small GTPases has been implicated in plexin signaling cascades through its RasGAP domain. However, little is known about how Ras family GTPases are controlled in vivo by plexin signaling. Here, we found that Drosophila Rap1, a member of the Ras family of GTPases, plays an important role controlling intersegmental nerve b motor axon guidance during neural development. Gain-of-function studies using dominant-negative and constitutively active forms of Rap1 indicate that Rap1 contributes to axonal growth and guidance. Genetic interaction analyses demonstrate that the Sema-1a/PlexA-mediated repulsive guidance function is regulated positively by Rap1. Furthermore, neuronal expression of mutant PlexA robustly restored defasciculation defects in PlexA null mutants when the catalytic arginine fingers of the PlexA RasGAP domain critical for GAP activity were disrupted. However, deleting the RasGAP domain abolished the ability of PlexA to rescue the PlexA guidance phenotypes. These findings suggest that PlexA-mediated motor axon guidance is dependent on the presence of the PlexA RasGAP domain, but not on its GAP activity toward Ras family small GTPases.

The Control of semaphorin-1a-mediated reverse signaling by opposing pebble and RhoGAPp190 functions in drosophila.

Transmembrane semaphorins (Semas) serve evolutionarily conserved guidance roles, and some function as both ligands and receptors. However, the molecular mechanisms underlying the transduction of these signals to the cytoskeleton remain largely unknown. We have identified two direct regulators of Rho family small GTPases, pebble (a Rho guanine nucleotide exchange factor [GEF]) and RhoGAPp190 (a GTPase activating protein [GAP]), that show robust interactions with the cytoplasmic domain of the Drosophila Sema-1a protein. Neuronal pebble and RhoGAPp190 are required to control motor axon defasciculation at specific pathway choice points and also for target recognition during Drosophila neuromuscular development. Sema-1a-mediated motor axon defasciculation is promoted by pebble and inhibited by RhoGAPp190. Genetic analyses show that opposing pebble and RhoGAPp190 functions mediate Sema-1a reverse signaling through the regulation of Rho1 activity. Therefore, pebble and RhoGAPp190 transduce transmembrane semaphorin-mediated guidance cue information that regulates the establishment of neuronal connectivity during Drosophila development.

Evolution of the tan locus contributed to pigment loss in Drosophila santomea: a response to Matute et al.

We have shown previously that the loss of abdominal pigmentation in D. santomea relative to its sister species D. yakuba resulted, in part, from cis-regulatory mutations at the tan locus. Matute et al. claim, based solely upon extrapolation from genetic crosses of D. santomea and D. melanogaster, a much more divergent species, that at least four X chromosome regions but not tan are responsible for pigmentation differences. Here, we provide additional evidence from introgressions of D. yakuba genes into D. santomea that support a causative role for tan in the loss of pigmentation and present analyses that contradict Matute et al.'s claims. We discuss how the choice of parental species and other factors affect the ability to identify loci responsible for species divergence, and we affirm that all of our previously reported results and conclusions stand.

The evolution of gene regulation underlies a morphological difference between two Drosophila sister species.

Understanding the mechanisms underlying the morphological divergence of species is one of the central goals of evolutionary biology. Here, we analyze the genetic and molecular bases of the divergence of body pigmentation patterns between Drosophila yakuba and its sister species Drosophila santomea. We found that loss of pigmentation in D. santomea involved the selective loss of expression of the tan and yellow pigmentation genes. We demonstrate that tan gene expression was eliminated through the mutational inactivation of one specific tan cis-regulatory element (CRE) whereas the Tan protein sequence remained unchanged. Surprisingly, we identify three independent loss-of-function alleles of the tan CRE in the young D. santomea lineage. We submit that there is sufficient empirical evidence to support the general prediction that functional evolutionary changes at pleiotropic loci will most often involve mutations in their discrete, modular cis-regulatory elements.