A neuroendocrine pathway modulating osmotic stress in Drosophila.

Environmental factors challenge the physiological homeostasis in animals, thereby evoking stress responses. Various mechanisms have evolved to counter stress at the organism level, including regulation by neuropeptides. In recent years, much progress has been made on the mechanisms and neuropeptides that regulate responses to metabolic/nutritional stress, as well as those involved in countering osmotic and ionic stresses. Here, we identified a peptidergic pathway that links these types of regulatory functions. We uncover the neuropeptide Corazonin (Crz), previously implicated in responses to metabolic stress, as a neuroendocrine factor that inhibits the release of a diuretic hormone, CAPA, and thereby modulates the tolerance to osmotic and ionic stress. Both knockdown of Crz and acute injections of Crz peptide impact desiccation tolerance and recovery from chill-coma. Mapping of the Crz receptor (CrzR) expression identified three pairs of Capa-expressing neurons (Va neurons) in the ventral nerve cord that mediate these effects of Crz. We show that Crz acts to restore water/ion homeostasis by inhibiting release of CAPA neuropeptides via inhibition of cAMP production in Va neurons. Knockdown of CrzR in Va neurons affects CAPA signaling, and consequently increases tolerance for desiccation, ionic stress and starvation, but delays chill-coma recovery. Optogenetic activation of Va neurons stimulates excretion and simultaneous activation of Crz and CAPA-expressing neurons reduces this response, supporting the inhibitory action of Crz. Thus, Crz inhibits Va neurons to maintain osmotic and ionic homeostasis, which in turn affects stress tolerance. Earlier work demonstrated that systemic Crz signaling restores nutrient levels by promoting food search and feeding. Here we additionally propose that Crz signaling also ensures osmotic homeostasis by inhibiting release of CAPA neuropeptides and suppressing diuresis. Thus, Crz ameliorates stress-associated physiology through systemic modulation of both peptidergic neurosecretory cells and the fat body in Drosophila.

HCN Channel Activity Balances Quiescence and Proliferation in Neural Stem Cells and Is a Selective Target for Neuroprotection During Cancer Treatment.

Children suffering from neurologic cancers undergoing chemotherapy and radiotherapy are at high risk of reduced neurocognitive abilities likely via damage to proliferating neural stem cells (NSC). Therefore, strategies to protect NSCs are needed. We argue that induced cell-cycle arrest/quiescence in NSCs during cancer treatment can represent such a strategy. Here, we show that hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels are dynamically expressed over the cell cycle in NSCs, depolarize the membrane potential, underlie spontaneous calcium oscillations and are required to maintain NSCs in the actively proliferating pool. Hyperpolarizing pharmacologic inhibition of HCN channels during exposure to ionizing radiation protects NSCs cells in neurogenic brain regions of young mice. In contrast, brain tumor-initiating cells, which also express HCN channels, remain proliferative during HCN inhibition. IMPLICATIONS: Our finding that NSCs can be selectively rescued while cancer cells remain sensitive to the treatment, provide a foundation for reduction of cognitive impairment in children with neurologic cancers.

Comparative cell cycle transcriptomics reveals synchronization of developmental transcription factor networks in cancer cells.

The cell cycle coordinates core functions such as replication and cell division. However, cell-cycle-regulated transcription in the control of non-core functions, such as cell identity maintenance through specific transcription factors (TFs) and signalling pathways remains unclear. Here, we provide a resource consisting of mapped transcriptomes in unsynchronized HeLa and U2OS cancer cells sorted for cell cycle phase by Fucci reporter expression. We developed a novel algorithm for data analysis that enables efficient visualization and data comparisons and identified cell cycle synchronization of Notch signalling and TFs associated with development. Furthermore, the cell cycle synchronizes with the circadian clock, providing a possible link between developmental transcriptional networks and the cell cycle. In conclusion we find that cell cycle synchronized transcriptional patterns are temporally compartmentalized and more complex than previously anticipated, involving genes, which control cell identity and development.

Peptidergic clock neurons in Drosophila: ion transport peptide and short neuropeptide F in subsets of dorsal and ventral lateral neurons.

About 150 clock neurons are clustered in different groups in the brain of Drosophila. Among these clock neurons, some pigment-dispersing factor (PDF)-positive and PDF-negative lateral neurons (LNs) are principal oscillators responsible for bouts of activity in the morning and evening, respectively. The full complement of neurotransmitters in these morning and evening oscillators is not known. By using a screen for candidate neuromediators in clock neurons, we discovered ion transport peptide (ITP) and short neuropeptide F (sNPF) as novel neuropeptides in subpopulations of dorsal (LN(d)s) and ventral (s-LN(v)s) LNs. Among the six LN(d)s, ITP was found in one that coexpresses long neuropeptide F (NPF) and cryptochrome. We detected sNPF in two LN(d)s that also express cryptochrome; these cells are distinct from three LN(d)s expressing NPF. Thus, we have identified neuropeptides in five of the six LN(d)s. The three LN(d)s expressing cryptochrome, with either ITP or sNPF, are the only ones with additional projections to the accessory medulla. Among the five s-LN(v)s in the adult brain, ITP was detected in the fifth neuron that is devoid of PDF and sNPF in the four neurons that also express PDF. By using a choline acetyltransferase (Cha) Gal4, we detected Cha expression in the two sNPF producing LN(d)s and in the fifth s-LN(v). In the larval brain, two of the four PDF-producing s-LN(v)s coexpress sNPF. Our findings emphasize that the LN(d)s are heterogeneous both anatomically and with respect to content of neuropeptides, cryptochrome, and other markers and suggest diverse functions of these neurons.

A large population of diverse neurons in the Drosophila central nervous system expresses short neuropeptide F, suggesting multiple distributed peptide functions.

BACKGROUND: Insect neuropeptides are distributed in stereotypic sets of neurons that commonly constitute a small fraction of the total number of neurons. However, some neuropeptide genes are expressed in larger numbers of neurons of diverse types suggesting that they are involved in a greater diversity of functions. One of these widely expressed genes, snpf, encodes the precursor of short neuropeptide F (sNPF). To unravel possible functional diversity we have mapped the distribution of transcript of the snpf gene and its peptide products in the central nervous system (CNS) of Drosophila in relation to other neuronal markers. RESULTS: There are several hundreds of neurons in the larval CNS and several thousands in the adult Drosophila brain expressing snpf transcript and sNPF peptide. Most of these neurons are intrinsic interneurons of the mushroom bodies. Additionally, sNPF is expressed in numerous small interneurons of the CNS, olfactory receptor neurons (ORNs) of the antennae, and in a small set of possibly neurosecretory cells innervating the corpora cardiaca and aorta. A sNPF-Gal4 line confirms most of the expression pattern. None of the sNPF immunoreactive neurons co-express a marker for the transcription factor DIMMED, suggesting that the majority are not neurosecretory cells or large interneurons involved in episodic bulk transmission. Instead a portion of the sNPF producing neurons co-express markers for classical neurotransmitters such as acetylcholine, GABA and glutamate, suggesting that sNPF is a co-transmitter or local neuromodulator in ORNs and many interneurons. Interestingly, sNPF is coexpressed both with presumed excitatory and inhibitory neurotransmitters. A few sNPF expressing neurons in the brain colocalize the peptide corazonin and a pair of dorsal neurons in the first abdominal neuromere coexpresses sNPF and insulin-like peptide 7 (ILP7). CONCLUSION: It is likely that sNPF has multiple functions as neurohormone as well as local neuromodulator/co-transmitter in various CNS circuits, including olfactory circuits both at the level of the first synapse and at the mushroom body output level. Some of the sNPF immunoreactive axons terminate in close proximity to neurosecretory cells producing ILPs and adipokinetic hormone, indicating that sNPF also might regulate hormone production or release.

Intrinsic neurons of Drosophila mushroom bodies express short neuropeptide F: relations to extrinsic neurons expressing different neurotransmitters.

Mushroom bodies constitute prominent paired neuropils in the brain of insects, known to be involved in higher olfactory processing and learning and memory. In Drosophila there are about 2,500 intrinsic mushroom body neurons, Kenyon cells, and a large number of different extrinsic neurons connecting the calyx, peduncle, and lobes to other portions of the brain. The neurotransmitter of the Kenyon cells has not been identified in any insect. Here we show expression of the gene snpf and its neuropeptide products (short neuropeptide F; sNPFs) in larval and adult Drosophila Kenyon cells by means of in situ hybridization and antisera against sequences of the precursor and two of the encoded peptides. Immunocytochemistry displays peptide in intrinsic neuronal processes in most parts of the mushroom body structures, except for a small core in the center of the peduncle and lobes and in the alpha'- and beta'-lobes. Weaker immunolabeling is seen in Kenyon cell bodies and processes in the calyx and initial peduncle and is strongest in the more distal portions of the lobes. We used different antisera and Gal4-driven green fluorescent protein to identify Kenyon cells and different populations of extrinsic neurons defined by their signal substances. Thus, we display neurotransmitter systems converging on Kenyon cells: neurons likely to utilize dopamine, tyramine/octopamine, glutamate, and acetylcholine. Attempts to identify other neurotransmitter components (including vesicular glutamate transporter) in Kenyon cells failed. However, it is likely that the Kenyon cells utilize an additional neurotransmitter, yet to be identified, and that the neuropeptides described here may represent cotransmitters.

Single-cell peptidomics of drosophila melanogaster neurons identified by Gal4-driven fluorescence.

Neuropeptides are widespread signal molecules that display a great chemical and functional diversity. Predictions of neuropeptide cleavage from precursor proteins are not always correct, and thus, biochemical identification is essential. Single-cell analysis is valuable to identify peptides processed from a single precursor, but also to determine coexpression of further neuropeptides from other precursors. We have developed an approach to isolate single identified neurons from the fruit fly Drosophila melanogaster for mass spectrometric analysis. By using Gal4 promoter lines to drive green fluorescent protein under UAS control, we identified specific peptidergic neurons. These neurons were isolated singly under a fluorescence microscope and subjected to MALDI-TOF mass spectrometry. Two Gal4 lines were used here to identify pigment-dispersing factor (PDF) and hugin-expressing neurons. We found that the large PDF expressing clock neurons only give rise to a single peptide, PDF. The three different classes of hugin expressing neurons all display the same mass signal, identical to pyrokinin-2. The other peptide predicted from the hugin precursor, hugin gamma, was not detected in any of the cells. Single-cell peptidomics is a powerful tool in Drosophila neuroscience since Gal4 drivers can be produced for all known neuropeptide genes and thus provide detailed information about neuropeptide complements in neurons of interest.

Diuretic action of the peptide locustatachykinin I: cellular localisation and effects on fluid secretion in Malpighian tubules of locusts.

In insects primary urine is produced by the Malpighian tubules under hormonal control. Here we have analysed the effects of the peptide locustatachykinin I (Lom-TK-I) on secretion in isolated Malphigian tubules. We also mapped the distribution of Lom-TK immunoreactivity in the gut in comparison with Locusta diuretic hormone (Lom-DH) and serotonin, two other factors that are active on locust tubules. Lom-TK-I produces an immediate, potent and long-lasting stimulation of fluid secretion. Furthermore, we show that Lom-TK-I acts synergistically with Lom-DH on fluid secretion and demonstrate that Lom-TKs are co-localised with Lom-DH in endocrine cells of the midgut ampullae. Thus, the two peptides might be released together to act synergistically on fluid secretion. Also serotonin and Lom-DH act synergistically and we can demonstrate a plexus of serotonin-containing axon processes over the midgut.