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 Aβ, polyglutamine, and Huntingtin. Of 219 C. elegans orthologs, knockdown of 16 enhanced both Aβ 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.
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•Chaperome expression is dramatically affected in human brain aging•Chaperome dynamics correlate with aging and neurodegenerative disease•Functional analysis identifies a chaperome subset that safeguards proteostasis
Brehme et al. have examined the chaperome from C. elegans to humans using functional assays and expression as well as protein-interactome analysis. The authors identify a conserved C. elegans chaperome subnetwork of 16 chaperone genes, corresponding to 28 human orthologs that are affected in brain aging and diseases associated with protein aggregation.
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.
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.
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.
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.
Sexual orientation and courtship behavior in Drosophila are regulated by
fruitless (fru), the first gene in a branch of the sex-determination hierarchy functioning specifically in the central nervous ...system (CNS). The phenotypes of new
fru mutants encompass nearly all aspects of male sexual behavior. Alternative splicing of
fru transcripts produces sex-specific proteins belonging to the BTB-ZF family of transcriptional regulators. The sex-specific
fru products are produced in only about 500 of the 10
5 neurons that comprise the CNS. The properties of neurons expressing these
fru products suggest that
fru specifies the fates or activities of neurons that carry out higher order control functions to elicit and coordinate the activities comprising male courtship behavior.
A multibranched hierarchy of regulatory genes controls all aspects of somatic sexual development in Drosophila melanogaster. One branch of this hierarchy is headed by the fruitless (fru) gene and ...functions in the central nervous system, where it is necessary for male courtship behavior as well as the differentiation of a male-specific abdominal structure, the muscle of Lawrence (MOL). A preliminary investigation of several of the mutations described here showed that the fru gene also has a sex-nonspecific vital function. The fru gene produces a complex set of transcripts through the use of four promoters and alternative splicing. Only the primary transcripts produced from the most distal (P1) promoter are sex-specifically spliced under direction of the sex-determination hierarchy. We have analyzed eight new fru mutations, created by X-ray mutagenesis and P-element excision, to try to gain insight into the relationship of specific transcript classes to specific fru functions. Males that lack the P1-derived fru transcripts show a complete absence of sexual behavior, but no other defects besides the loss of the MOL. Both males and females that have reduced levels of transcripts from the P3 promoter develop into adults but frequently die after failing to eclose. Analysis of the morphology and behavior of adult escapers showed that P3-encoded functions are required for the proper differentiation and eversion of imaginal discs. Furthermore, the reduction in the size of the neuromuscular junctions on abdominal muscles in these animals suggests that one of fru's sex-nonspecific functions involves general aspects of neuronal differentiation. In mutants that lack all fru transcripts as well as a small number of adjacent genes, animals die at an early pupal stage, indicating that fru's function is required only during late development. Thus, fru functions both in the sex-determination regulatory hierarchy to control male sexual behavior through sex-specific transcripts and sex-nonspecifically to control the development of imaginal discs and motorneuronal synapses during adult development through sex-nonspecific transcript classes.
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.
The fruitless mutants fru3 and fru4 were assessed for sex-specific reproductive-behavioral phenotypes and compared to the previously reported fru mutants. Among the several behavioral anomalies ...exhibited by males expressing these relatively new mutations, some are unique. fru3 and fru4 males are less stimulated to court females than fru1 and fru2. No courtship pulse song is generated by either fru3 or fru4 males, even though they perform brief wing extensions. fru3 and fru4 males display significantly less chaining behavior than do fru1 males. The hierarchy of courtship responses by fru males directed toward females vs. males, when presented with both sexes simultaneously, is that fru1 males perform vigorous and indiscriminant courtship directed at either sex; fru4 males are similarly indiscriminant, but courtship levels were lower than fru1; fru2 males prefer females; fru3 males show a courtship bias toward males. fru3 and fru4 males essentially lack the Muscle of Lawrence (MOL). On several reproductive criteria, there was no difference between fru-variant females and fru+. The increases in phenotypic severity measured for the new mutants are discussed in the context of the emerging molecular genetics of fru and with regard to the gene's position within the sex-determination pathway.
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.
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 (shiTS). We extended these manipulations to analyze all features of male-specific behavior, including courtship song, which was almost eliminated by driving shiTS 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/traF 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 FRUM protein is regulated by TRA, we hypothesized that a fru-derived transgene encoding the male (M) form of an Inhibitory RNA (fruMIR) would mimic the effects of traF; but C309/fruMIR 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 FRUM in wild-type males included coexpressing neurons. Histological analysis of the developing CNS could not rationalize the absence of traF or fruMIR effects on courtship song, because we found C309 to be coexpressed with FRUM within the same 10 neuronal clusters in pupae. Thus, we hypothesize that elimination of singing behavior by the C309/shiTS combination involves neurons acting downstream of FRUM cells
The role played by the sex-determining gene doublesex (dsx) and its influence on Drosophila courtship were examined. Against a background of subnormal male-like behavior that is reported to be an ...attribute of haplo-X flies homozygous for the original dsx mutation, and given that a sex-specific muscle is unaffected by genetic variation at this locus, analyses of several reproductive behaviors and control for genetic background effects indicated that XY dsx mutants are impaired in their willingness to court females. When they did court, certain behavioral actions were normal, including components of courtship song. However, these mutants never produced courtship humming sounds. Mature XY dsx flies elicited anomalously high levels of courtship; that this occurs merely because of a delay in imaginal development was experimentally discounted. The current analysis reconciled two ostensibly conflicting reports involving the courtship-stimulating qualities of this mutant type. Such experiments also uncovered a new behavioral anomaly: dsx mutations caused chromosomal males to court other males at abnormally high levels. These results are discussed from the perspective of doublesex's influence on internal tissues of adult Drosophila involved in the triggering and neural control of male- and female-like elements of courtship, reproductive pheromone production, or a combination of such factors
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