Firefly luciferase mutants as sensors of proteome stress.

Maintenance of cellular protein homeostasis (proteostasis) depends on a complex network of molecular chaperones, proteases and other regulatory factors. Proteostasis deficiency develops during normal aging and predisposes individuals for many diseases, including neurodegenerative disorders. Here we describe sensor proteins for the comparative measurement of proteostasis capacity in different cell types and model organisms. These sensors are increasingly structurally destabilized versions of firefly luciferase. Imbalances in proteostasis manifest as changes in sensor solubility and luminescence activity. We used EGFP-tagged constructs to monitor the aggregation state of the sensors and the ability of cells to solubilize or degrade the aggregated proteins. A set of three sensor proteins serves as a convenient toolkit to assess the proteostasis status in a wide range of experimental systems, including cell and organism models of stress, neurodegenerative disease and aging.

Male-specific fruitless specifies the neural substrates of Drosophila courtship behaviour.

Robust innate behaviours are attractive systems for genetically dissecting how environmental cues are perceived and integrated to generate complex behaviours. During courtship, Drosophila males engage in a series of innate, stereotyped behaviours that are coordinated by specific sensory cues. However, little is known about the specific neural substrates mediating this complex behavioural programme. Genetic, developmental and behavioural studies have shown that the fruitless (fru) gene encodes a set of male-specific transcription factors (FruM) that act to establish the potential for courtship in Drosophila. FruM proteins are expressed in approximately 2% of central nervous system neurons, at least one subset of which coordinates the component behaviours of courtship. Here we have inserted the yeast GAL4 gene into the fru locus by homologous recombination and show that (1) FruM is expressed in subsets of all peripheral sensory systems previously implicated in courtship, (2) inhibition of FruM function in olfactory system components reduces olfactory-dependent changes in courtship behaviour, (3) transient inactivation of all FruM-expressing neurons abolishes courtship behaviour, with no other gross changes in general behaviour, and (4) 'masculinization' of FruM-expressing neurons in females is largely sufficient to confer male courtship behaviour. Together, these data demonstrate that FruM proteins specify the neural substrates of male courtship.

Amplifiers co-translationally enhance CFTR biosynthesis via PCBP1-mediated regulation of CFTR mRNA.

BACKGROUND: Cystic fibrosis (CF) is a recessive disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. We previously described a first-in-class CFTR modulator that functions as an amplifier to selectively increase CFTR expression and function. The amplifier mechanism is distinct from and complementary to corrector and potentiator classes of CFTR modulators. Here we characterize the mechanism by which amplifiers increase CFTR mRNA, protein, and activity. METHODS: Biochemical studies elucidated the action of amplifiers on CFTR mRNA abundance and translation and defined the role of an amplifier-binding protein that was identified using chemical proteomics. RESULTS: Amplifiers stabilize CFTR mRNA through a process that requires only the translated sequence of CFTR and involves translational elongation. Amplifiers enrich ER-associated CFTR mRNA and increase its translational efficiency through increasing the fraction of CFTR mRNA associated with polysomes. Pulldowns identified the poly(rC)-binding protein 1 (PCBP1) as directly binding to amplifier. A PCBP1 consensus element was identified within the CFTR open reading frame that binds PCBP1. This sequence proved necessary for amplifier responsiveness. CONCLUSIONS: Small molecule amplifiers co-translationally increase CFTR mRNA stability. They enhance translation through addressing the inherently inefficient membrane targeting of CFTR mRNA. Amplifiers bind directly to PCBP1, show enhanced affinity in the presence of bound RNA, and require a PCBP1 consensus element within CFTR mRNA to elicit translational effects. These modulators represent a promising new and mechanistically novel class of CFTR therapeutic. They may be useful as a monotherapy or in combination with other CFTR modulators.

Isoform-specific control of male neuronal differentiation and behavior in Drosophila by the fruitless gene.

BACKGROUND: How the central nervous system (CNS) develops to implement innate behaviors remains largely unknown. Drosophila male sexual behavior has long been used as a model to address this question. The male-specific products of fruitless (fru) are pivotal to the emergence of this behavior. These putative transcription factors, containing one of three alternative DNA binding domains, determine the neuronal substrates for sexual behavior in male CNS. RESULTS: We isolated the first fru coding mutation, resulting in complete loss of one isoform. At the neuronal level, this isoform alone controls differentiation of a male-specific muscle and its associated motorneuron. Conversely, a combination of isoforms is required for development of serotonergic neurons implicated in male copulatory behavior. Full development of these neurons requires the male-specific product of doublesex, a gene previously thought to act independently of fru. At the behavioral level, missing one isoform leads to diminished courtship behavior and infertility. We achieved the first rescue of a distinct fru behavioral phenotype, expressing a wild-type isoform in a defined subset of its normal expression pattern. CONCLUSION: This study exemplifies how complex behaviors can be controlled by a single locus through multiple isoforms regulating both developmental and physiological pathways in different neuronal substrates.

fruitless Gene products truncated of their male-like qualities promote neural and behavioral maleness in Drosophila if these proteins are produced in the right places at the right times.

To bring GAL4 production under the control of the sex promoter (P1) contained within Drosophila's fruitless gene, a gal4 cassette was previously inserted downstream of P1. This insert should eliminate male-specific FRU(M) proteins, which normally contain 101 amino acids (aa's) at their N termini. Thus males homozygous for the P1-gal4 insert should be courtless, as was briefly stated to be so in the initial report of this transgenic type. But XY flies whose only fru form is P1-gal4 have now been found to court vigorously. P1-gal4 females displayed no appreciable male-like actions except courtship rejection behaviors; yet, they developed a male-specific abdominal muscle. No immunoreactivity against the male-specific aa's was detectable in P1-gal4 flies. But male-like neural signals were observed in XY or XX P1-gal4 pupae and adults after applying an antibody that detects all FRU isoforms; transgenic females displayed reduced expression of such proteins. RT-PCR's rationalized these findings: P1 transcripts include anomalous splice forms from which gal4 was removed, allowing FRU's lacking M aa's to be produced in male-like patterns in both sexes. Within males, such defective proteins promote neural differentiation and function that is sufficient to support spirited P1-gal4 courtship. But dispensability of the male-specific FRU N-terminus is tempered by the finding that intra-fru sequences encoding these 101 aa's are highly conserved among interspecific relatives of D. melanogaster.

Orkambi® and amplifier co-therapy improves function from a rare CFTR mutation in gene-edited cells and patient tissue.

The combination therapy of lumacaftor and ivacaftor (Orkambi®) is approved for patients bearing the major cystic fibrosis (CF) mutation: ΔF508 It has been predicted that Orkambi® could treat patients with rarer mutations of similar "theratype"; however, a standardized approach confirming efficacy in these cohorts has not been reported. Here, we demonstrate that patients bearing the rare mutation: c.3700 A>G, causing protein misprocessing and altered channel function-similar to ΔF508-CFTR, are unlikely to yield a robust Orkambi® response. While in silico and biochemical studies confirmed that this mutation could be corrected and potentiated by lumacaftor and ivacaftor, respectively, this combination led to a minor in vitro response in patient-derived tissue. A CRISPR/Cas9-edited bronchial epithelial cell line bearing this mutation enabled studies showing that an "amplifier" compound, effective in increasing the levels of immature CFTR protein, augmented the Orkambi® response. Importantly, this "amplifier" effect was recapitulated in patient-derived nasal cultures-providing the first evidence for its efficacy in augmenting Orkambi® in tissues harboring a rare CF-causing mutation. We propose that this multi-disciplinary approach, including creation of CRISPR/Cas9-edited cells to profile modulators together with validation using primary tissue, will facilitate therapy development for patients with rare CF mutations.

Courtship and other behaviors affected by a heat-sensitive, molecularly novel mutation in the cacophony calcium-channel gene of Drosophila.

The cacophony (cac) locus of Drosophila melanogaster, which encodes a calcium-channel subunit, has been mutated to cause courtship-song defects or abnormal responses to visual stimuli. However, the most recently isolated cac mutant was identified as an enhancer of a comatose mutation's effects on general locomotion. We analyzed the cac(TS2) mutation in terms of its intragenic molecular change and its effects on behaviors more complex than the fly's elementary ability to move. The molecular etiology of this mutation is a nucleotide substitution that causes a proline-to-serine change in a region of the polypeptide near its EF hand. Given that this motif is involved in channel inactivation, it was intriguing that cac(TS2) males generate song pulses containing larger-than-normal numbers of cycles--provided that such males are exposed to an elevated temperature. Similar treatments caused only mild visual-response abnormalities and generic locomotor sluggishness. These results are discussed in the context of calcium-channel functions that subserve certain behaviors and of defects exhibited by the original cacophony mutant. Despite its different kind of amino-acid substitution, compared with that of cac(TS2), cac(S) males sing abnormally in a manner that mimics the new mutant's heat-sensitive song anomaly.

A chaperome subnetwork safeguards proteostasis in aging and neurodegenerative disease.

Chaperones are central to the proteostasis network (PN) and safeguard the proteome from misfolding, aggregation, and proteotoxicity. We categorized the human chaperome of 332 genes into network communities using function, localization, interactome, and expression data sets. During human brain aging, expression of 32% of the chaperome, corresponding to ATP-dependent chaperone machines, is repressed, whereas 19.5%, corresponding to ATP-independent chaperones and co-chaperones, are induced. These repression and induction clusters are enhanced in the brains of those with Alzheimer's, Huntington's, or Parkinson's disease. Functional properties of the chaperome were assessed by perturbation in C. elegans and human cell models expressing Aß, polyglutamine, and Huntingtin. Of 219 C. elegans orthologs, knockdown of 16 enhanced both Aß and polyQ-associated toxicity. These correspond to 28 human orthologs, of which 52% and 41% are repressed, respectively, in brain aging and disease and 37.5% affected Huntingtin aggregation in human cells. These results identify a critical chaperome subnetwork that functions in aging and disease.

Neurogenetics of courtship and mating in Drosophila.

The reproductive biology of Drosophila melanogaster is described and critically discussed, primarily with regard to genetic studies of sex-specific behavior and its neural underpinnings. The investigatory history of this system includes, in addition to a host of recent neurobiological analyses of reproductive phenotypes, studies of mating as well as the behaviors leading up to that event. Courtship and mating have been delved into mostly with regard to male-specific behavior and biology, although a small number of studies has also pointed to the neural substrates of female reproduction. Sensory influences on interactions between courting flies have long been studied, partly by application of mutants and partly by surgical experiments. More recently, molecular-genetic approaches to sensations passing between flies in reproductive contexts have aimed to "dissect" further the meaning of separate sensory modalities. Notable among these are olfactory and contact-chemosensory stimuli, which perhaps have received an inordinate amount of attention in terms of the possibility that they could comprise the key cues involved in triggering and sustaining courtship actions. But visual and auditory stimuli are heavily involved as well--appreciated mainly from older experiments, but analyzable further using elementary approaches (single-gene mutations mutants and surgeries), as well as by applying the molecularly defined factors alluded to above. Regarding regulation of reproductive behavior by components of Drosophila's central nervous system (CNS), once again significant invigoration of the relevant inquiries has been stimulated and propelled by identification and application of molecular-genetic materials. A distinct plurality of the tools applied involves transposons inserted in the fly's chromosomes, defining "enhancer-trap" strains that can be used to label various portions of the nervous system and, in parallel, disrupt their structure and function by "driving" companion transgenes predesigned for these experimental purposes. Thus, certain components of interneuronal routes, functioning along pathways whose starting points are sensory reception by the peripheral nervous system (PNS), have been manipulated to enhance appreciation of sexually important sensory modalities, as well as to promote understanding of where such inputs end up within the CNS: Where are reproductively related stimuli processed, such that different kinds of sensation would putatively be integrated to mediate sex-specific behavioral readouts? In line with generic sensory studies that have tended to concentrate on chemical stimuli, PNS-to-CNS pathways focused upon in reproductive experiments relying on genic enhancers have mostly involved smell and taste. Enhancer traps have also been applied to disrupt various regions within the CNS to ask about the various ganglia, and portions thereof, that contribute to male- or female-specific behavior. These manipulations have encompassed structural or functional disruptions of such regions as well as application of molecular-genetic tricks to feminize or masculinize a given component of the CNS. Results of such experiments have, indeed, identified certain discrete subsets of centrally located ganglia that, on the one hand, lead to courtship defects when disrupted or, on the other, must apparently maintain sex-specific identity if the requisite courtship actions are to be performed. As just implied, perturbations of certain neural tissues not based on manipulating "sex factors" might lead to reproductive behavioral abnormalities, even though changing the sexual identity of such structures would not necessarily have analogous consequences. It has been valuable to uncover these sexually significant subsets of the Drosophila nervous system, although it must be said that not all of the transgenically based dissection outcomes are in agreement. Thus, the good news is that not all of the CNS is devoted to courtship control, whereby any and all locales disrupted might have led to sex-specific deficits; but the bad news is that the enhancer-trap approach to these matters has not led to definitive homing-in on some tractable number of mutually agreed-upon "courtship centers" within the brain or within the ventral nerve cord (VNC). The latter neural region, which comprises about half of the fly's CNS, is underanalyzed as to its sex-specific significance: How, for example, are various kinds of sensory inputs to posteriorly located PNS structures processed, such that they eventually end up modulating brain functions underlying courtship? And how are sex-specific motor outputs mediated by discrete collections of neurons within VNC ganglia--so that, for instance, male-specific whole-animal motor actions and appendage usages are evoked? These behaviors can be thought of as fixed action patterns. But it is increasingly appreciated that elements of the fly's reproductive behavior can be modulated by previous experience. In this regard, the neural substrates of conditioned courtship are being more and more analyzed, principally by further usages of various transgenic types. Additionally, a set of molecular neurogenetic experiments devoted to experience-dependent courtship was based on manipulations of a salient "sex gene" in D. melanogaster. This well-defined factor is called fruitless (fru). The gene, its encoded products, along with their behavioral and neurobiological significance, have become objects of frenetic attention in recent years. How normal, mutated, and molecularly manipulated forms of fru seem to be generating a good deal of knowledge and insight about male-specific courtship and mating is worthy of much attention. This previews the fact that fruitless matters are woven throughout this chapter as well as having a conspicuous section allocated to them. Finally, an acknowledgment that the reader is being subjected to lengthy preview of an article about this subject is given. This matter is mentioned because--in conjunction with the contemporary broadening and deepening of this investigatory area--brief summaries of its findings are appearing with increasing frequency. This chapter will, from time to time, present our opinion that a fair fraction of the recent minireviews are replete with too many catch phrases about what is really known. This is one reason why the treatment that follows not only attempts to describe the pertinent primary reports in detail but also pauses often to discuss our views about current understandings of sex-specific behavior in Drosophila and its underlying biology.

Impaired clock output by altered connectivity in the circadian network.

Substantial progress has been made in elucidating the molecular processes that impart a temporal control to physiology and behavior in most eukaryotes. In Drosophila, dorsal and ventral neuronal networks act in concert to convey rhythmicity. Recently, the hierarchical organization among the different circadian clusters has been addressed, but how molecular oscillations translate into rhythmic behavior remains unclear. The small ventral lateral neurons can synchronize certain dorsal oscillators likely through the release of pigment dispersing factor (PDF), a neuropeptide central to the control of rhythmic rest-activity cycles. In the present study, we have taken advantage of flies exhibiting a distinctive arrhythmic phenotype due to mutation of the potassium channel slowpoke (slo) to examine the relevance of specific neuronal populations involved in the circadian control of behavior. We show that altered neuronal function associated with the null mutation specifically impaired PDF accumulation in the dorsal protocerebrum and, in turn, desynchronized molecular oscillations in the dorsal clusters. However, molecular oscillations in the small ventral lateral neurons are properly running in the null mutant, indicating that slo is acting downstream of these core pacemaker cells, most likely in the output pathway. Surprisingly, disrupted PDF signaling by slo dysfunction directly affects the structure of the underlying circuit. Our observations demonstrate that subtle structural changes within the circadian network are responsible for behavioral arrhythmicity.

Defective transfer of seminal-fluid materials during matings of semi-fertile fruitless mutants in Drosophila.

In context of the semi-sterility exhibited by Drosophila males expressing certain mating-enabling fruitless (fru) mutant genotypes, we examined the transfer of seminal fluid using a transgene that encodes the Sex Peptide (SP) oligopeptide fused to Green Fluorescent Protein (GFP). We found that this fusion construct expresses SP-GFP in a valid manner within accessory glands of the male reproductive system in normal and fru-mutant males. Transfer of SP-GFP to live females was readily detectable during and after copulation. With respect to the pertinent combinations of fru mutations, we demonstrated that these abnormal genotypes cause males to transmit mating-related materials in two aberrant ways: one involving whether any seminal-fluid entities are transferred at all during a given mating; the other revealing an intriguing aspect of these fruitless effects, such that the mutations in question cause males to transfer female-affecting materials in a manner that varies among copulations. In this regard, certain mutant males that do not transfer SP nevertheless are able to transfer sperm: a fru-mated female possessing no GFP who was not fecund initially could produce progeny when seminal-fluid proteins were subsequently supplied by mating with a male that was spermless owing to the effects of a tudor mutation.

Neurotoxic protein expression reveals connections between the circadian clock and mating behavior in Drosophila.

To investigate the functions of circadian neurons, we added two strategies to the standard Drosophila behavioral genetics repertoire. The first was to express a polyglutamine-expanded neurotoxic protein (MJDtr78Q; MJD, Machado-Joseph disease) in the major timeless (tim)-expressing cells of the adult brain. These Tim-MJD flies were viable, in contrast to the use of cell-death gene expression for tim neuron inactivation. Moreover, they were more arrhythmic than flies expressing other neurotoxins and had low but detectable tim mRNA levels. The second extended standard microarray technology from fly heads to dissected fly brains. By combining the two approaches, we identified a population of Tim-MJD-affected mRNAs. Some had been previously identified as sex-specific and relevant to courtship, including mRNAs localized to brain-proximal fat-body tissue and brain courtship centers. Finally, we found a decrease in the number of neurons that expressed male-specific forms of the fruitless protein in the laterodorsal region of the brain. The decrease was not a consequence of toxic protein expression within these specialized cells but a likely effect of communication with neighboring TIM-expressing neurons. The data suggest a functional interaction between adjacent circadian and mating circuits within the fly brain, as well as an interaction between circadian circuits and brain-proximal fat body.

Functional analysis of fruitless gene expression by transgenic manipulations of Drosophila courtship.

A gal4-containing enhancer-trap called C309 was previously shown to cause subnormal courtship of Drosophila males toward females and courtship among males when driving a conditional disrupter of synaptic transmission (shi(TS)). We extended these manipulations to analyze all features of male-specific behavior, including courtship song, which was almost eliminated by driving shi(TS) at high temperature. In the context of singing defects and homosexual courtship affected by mutations in the fru gene, a tra-regulated component of the sex-determination hierarchy, we found a C309/tra(F) combination also to induce high levels of courtship between pairs of males and "chaining" behavior in groups; however, these doubly transgenic males sang normally. Because production of male-specific FRU(M) protein is regulated by TRA, we hypothesized that a fru-derived transgene encoding the male (M) form of an Inhibitory RNA (fru(MIR)) would mimic the effects of tra(F); but C309/fru(MIR) males exhibited no courtship chaining, although they courted other males in single-pair tests. Double-labeling of neurons in which GFP was driven by C309 revealed that 10 of the 20 CNS clusters containing FRU(M) in wild-type males included coexpressing neurons. Histological analysis of the developing CNS could not rationalize the absence of tra(F) or fru(MIR) effects on courtship song, because we found C309 to be coexpressed with FRU(M) within the same 10 neuronal clusters in pupae. Thus, we hypothesize that elimination of singing behavior by the C309/shi(TS) combination involves neurons acting downstream of FRU(M) cells.