@article {161983, title = {Synergism of IP3R and Parkin mutants identifies mitochondrial stress as an early feature of Parkinson{\textquoteright}s Disease.}, journal = {Dis Model Mech}, year = {2025}, month = {2025 Nov 14}, abstract = {

Our understanding of mechanisms underlying familial Parkinson{\textquoteright}s Disease (PD) have benefitted from studies in Drosophila models of PD. However, in a majority of PD patients the disease occurs sporadically and cellular phenotypes that arise early in sporadic PD remain to be understood. A genetic predisposition, arising from mutations in pathways that impact dopaminergic neuron health could be one cause of sporadic PD. Here, we studied Drosophila with single copies of recessive IP3R gene (itpr) mutants placed in combination with a recessive null mutant for the parkin gene. Whereas individual mutants appear normal, in combination they synergise to exhibit flight motor deficits with a focus in a subset of central dopaminergic neurons. Surprisingly, mitophagy and mitochondrial Ca2+ are barely affected. Instead, flight motor deficits correlate with elevated levels of mitochondrial H2O2 and reducing H2O2 levels by genetic means restored mitochondrial function and flight to a significant extent. This study underlines the importance of mitochondrial oxidative stress as an early phenotype in PD and suggests that humans with recessive mutations in either pathway have a higher chance of developing sporadic PD.

}, keywords = {Calcium homeostasis; Dopaminergic neurons; Flight; Motor function; PPL1}, issn = {1754-8411}, doi = {10.1242/dmm.052146}, author = {Dileep, Mrudula and Sharma, Anamika and Kasturacharya, Nandashree and Nizami, Syed Kavish and Manjila, Steffy and Bhan, Ashita and Hasan, Gaiti} } @article {140647, title = {IPRs and nSOCE-Tied Together at Two Ends.}, journal = {Contact (Thousand Oaks)}, volume = {7}, year = {2024}, month = {2024 Jan-Dec}, pages = {25152564241231092}, abstract = {

All living organisms need to respond appropriately to changes in the extracellular milieu. Cellular mechanisms that enable such responses evolved in parallel with organismal complexity and intracellular Ca signaling is one such mechanism where extracellular signals received at the cell membrane communicate with endoplasmic reticular stores of Ca, to stimulate appropriate Ca-mediated changes in cellular physiology. The amplitude and dynamics of endoplasmic reticulum (ER)-Ca release in response to extracellular signals determines the nature of the cellular response. An understanding of how ER-Ca channels might regulate cellular Ca signaling in different cell types is lacking. In a recent paper, this question has been addressed in the context of neurons ( Chakraborty et al., 2023) and the implications of these new findings are discussed here.

}, issn = {2515-2564}, doi = {10.1177/25152564241231092}, author = {Hasan, Gaiti} } @article {140644, title = {Orai-mediated calcium entry determines activity of central dopaminergic neurons by regulation of gene expression.}, journal = {Elife}, volume = {12}, year = {2024}, month = {2024 Jan 30}, abstract = {

Maturation and fine-tuning of neural circuits frequently require neuromodulatory signals that set the excitability threshold, neuronal connectivity, and synaptic strength. Here, we present a mechanistic study of how neuromodulator-stimulated intracellular Ca signals, through the store-operated Ca channel Orai, regulate intrinsic neuronal properties by control of developmental gene expression in flight-promoting central dopaminergic neurons (fpDANs). The fpDANs receive cholinergic inputs for release of dopamine at a central brain tripartite synapse that sustains flight (Sharma and Hasan, 2020). Cholinergic inputs act on the muscarinic acetylcholine receptor to stimulate intracellular Ca release through the endoplasmic reticulum (ER) localised inositol 1,4,5-trisphosphate receptor followed by ER-store depletion and Orai-mediated store-operated Ca entry (SOCE). Analysis of gene expression in fpDANs followed by genetic, cellular, and molecular studies identified Orai-mediated Ca entry as a key regulator of excitability in fpDANs during circuit maturation. SOCE activates the transcription factor trithorax-like (Trl), which in turn drives expression of a set of genes, including , that encodes a histone 3 lysine 36 methyltransferase (H3K36me3). Set2 function establishes a positive feedback loop, essential for receiving neuromodulatory cholinergic inputs and sustaining SOCE. Chromatin-modifying activity of Set2 changes the epigenetic status of fpDANs and drives expression of key ion channel and signalling genes that determine fpDAN activity. Loss of activity reduces the axonal arborisation of fpDANs within the MB lobe and prevents dopamine release required for the maintenance of long flight.

}, keywords = {Calcium, Calcium, Dietary, Cholinergic Agents, Dopamine, Dopaminergic Neurons, Histone-Lysine N-Methyltransferase}, issn = {2050-084X}, doi = {10.7554/eLife.88808}, author = {Mitra, Rishav and Richhariya, Shlesha and Hasan, Gaiti} } @article {123124, title = {ER-Ca2+ stores and the regulation of Store-operated Ca2+ Entry in neurons.}, journal = {J Physiol}, year = {2023}, month = {2023 Jan 24}, abstract = {

Key components of endoplasmic reticular (ER) Ca release and store-operated Ca entry (SOCE) are likely expressed in all metazoan cells. Due to the complexity of canonical Ca entry mechanisms in neurons, the functional significance of ER-Ca release and SOCE has been difficult to identify and establish. In this review we present evidence of how these two related mechanisms of Ca signaling impact multiple aspects of neuronal physiology and discuss their interaction with the better understood classes of ion channels that are gated by either voltage changes or extracellular ligands in neurons. Given how a small imbalance in Ca homeostasis can have strongly detrimental effects on neurons, leading to cell death, it is essential that neuronal SOCE is carefully regulated. We go onto discuss mechanisms of SOCE regulation identified in Drosophila and mammalian neurons. These include specific splice variants of STIM, different classes of membrane interacting proteins and an ER-Ca channel. So far these appear distinct from the mechanisms of SOCE regulation identified in non-excitable cells. Finally we touch upon the significance of these studies in the context of certain human neurodegenerative diseases. Abstract figure legend Store-operated Ca entry in neuronal cells is regulated by both positive (left) and negative regulators (right). This article is protected by copyright. All rights reserved.

}, issn = {1469-7793}, doi = {10.1113/JP283827}, author = {Chakraborty, Pragnya and Hasan, Gaiti} } @article {124444, title = {Generation of feeder-independent transgene-free iPSC lines from a young-onset Parkinson{\textquoteright}s disease (YOPD) patient with a homozygous PLA2G6: c.2222G>A (p. Arg741Gln) mutation (NCBSi003-A) and unaffected heterozygous parent (NCBSi004-A).}, journal = {Stem Cell Res}, volume = {67}, year = {2023}, month = {2023 Jan 24}, pages = {103033}, abstract = {

Phospholipase A2 group 6 (PLA2G6, iPLA2β or PARK14) gene encodes a calcium-independent group 6 phospholipase A2 enzyme and is associated with young-onset autosomal recessive Parkinson{\textquoteright}s disease (PD). We generated human induced pluripotent stem cell (iPSC) lines from a patient with young-onset PD carrying a homozygous PLA2G6: c.2222G\>A (p. Arg741Gln) mutation (NCBSi003-A) and unaffected heterozygous parent (NCBSi004-A). These iPSC lines will be used for investigating the key molecular signatures of young-onset PD (YOPD), and to understand the predictive phenotypes of the disease.

}, issn = {1876-7753}, doi = {10.1016/j.scr.2023.103033}, author = {Gopurappilly, Renjitha and Musthafa, Thasneem and Sukumaran, Salil and Viswanath, Biju and Hasan, Gaiti} } @article {131300, title = {Regulation of store-operated Ca2+ entry by IP3 receptors independent of their ability to release Ca2.}, journal = {Elife}, volume = {12}, year = {2023}, month = {07/2023}, abstract = {

Loss of endoplasmic reticular (ER) Ca2+ activates store-operated Ca2+ entry (SOCE) by causing the ER localized Ca2+ sensor STIM to unfurl domains that activate Orai channels in the plasma membrane at membrane contact sites (MCS). Here we demonstrate a novel mechanism by which the inositol 1,4,5 trisphosphate receptor (IP3R), an ER-localized IP3-gated Ca2+ channel, regulates neuronal SOCE. In human neurons, SOCE evoked by pharmacological depletion of ER-Ca2+ is attenuated by loss of IP3Rs, and restored by expression of IP3Rs even when they cannot release Ca2+, but only if the IP3Rs can bind IP3. Imaging studies demonstrate that IP3Rs enhance association of STIM1 with Orai1 in neuronal cells with empty stores; this requires an IP3-binding site, but not a pore. Convergent regulation by IP3Rs, may tune neuronal SOCE to respond selectively to receptors that generate IP3.

}, doi = {10.7554/eLife.80447}, author = {Chakraborty, P. and Deb, B. K. and Arige, V. and Musthafa, T. and Malik, S. and Yule, D. I. and Taylor, C.W. and Hasan, Gaiti} } @article {130543, title = {A STIM dependent dopamine-neuropeptide axis maintains the larval drive to feed and grow in Drosophila.}, journal = {PLoS Genet}, volume = {19}, year = {2023}, month = {2023 Jun 26}, pages = {e1010435}, abstract = {

Appropriate nutritional intake is essential for organismal survival. In holometabolous insects such as Drosophila melanogaster, the quality and quantity of food ingested as larvae determines adult size and fecundity. Here we have identified a subset of dopaminergic neurons (THD{\textquoteright}) that maintain the larval motivation to feed. Dopamine release from these neurons requires the ER Ca2+ sensor STIM. Larvae with loss of STIM stop feeding and growing, whereas expression of STIM in THD{\textquoteright} neurons rescues feeding, growth and viability of STIM null mutants to a significant extent. Moreover STIM is essential for maintaining excitability and release of dopamine from THD{\textquoteright} neurons. Optogenetic stimulation of THD{\textquoteright} neurons activated neuropeptidergic cells, including median neuro secretory cells that secrete insulin-like peptides. Loss of STIM in THD{\textquoteright} cells alters the developmental profile of specific insulin-like peptides including ilp3. Loss of ilp3 partially rescues STIM null mutants and inappropriate expression of ilp3 in larvae affects development and growth. In summary we have identified a novel STIM-dependent function of dopamine neurons that modulates developmental changes in larval feeding behaviour and growth.

}, issn = {1553-7404}, doi = {10.1371/journal.pgen.1010435}, author = {Kasturacharya, Nandashree and Dhall, Jasmine Kaur and Hasan, Gaiti} } @article {107012, title = {Store-operated Ca2+ entry regulates neuronal gene expression and function.}, journal = {Curr Opin Neurobiol}, volume = {73}, year = {2022}, month = {02/2022}, pages = {102520}, abstract = {

Recent studies with mutants of STIM and Orai have identified store-operated Ca2+ entry as an important regulator of neuronal function in Drosophila and mouse. Cellular Ca2+ imaging and electrophysiological studies demonstrate changes in ion channel function in neurons with loss of store-operated Ca2+ entry. Importantly, such changes are specific to neuronal subtypes. Transcriptomic and single-cell gene expression studies from the mouse brain identified wide, and isoform-specific differences, in expression of genes required for ER-store Ca2+ release and store-operated Ca2+ entry, across different neuronal classes. Loss of store-operated Ca2+ entry in neurons impacts neuronal gene expression profiles and includes genes encoding ion channels. The functional significance of store-operated Ca2+ entry across specific neuronal subtypes and in the context of neurodegenerative syndromes needs further study.

}, doi = {10.1016/j.conb.2022.01.005}, author = {Mitra, Rishav and Hasan, Gaiti} } @article {105898, title = {Two photon imaging of calcium responses in murine Purkinje neurons.}, journal = {STAR Protoc}, volume = {3}, year = {2022}, month = {2022 Mar 18}, pages = {101105}, abstract = {

Purkinje neurons (PNs) are an important component of the motor learning and coordination circuit and are affected in spino-cerebellar ataxias. Maintaining healthy PNs in cerebellar slices and recording their Ca transients can be challenging. Here, we describe a protocol for measuring Ca transients in PNs from adult mice, including problem-solving tips. This protocol can be used to measure neuronal excitability and agonist-mediated Ca signaling in cerebellar slices expressing a genetic Ca reporter in all PNs, thus improving yield of data. For complete details on the use and execution of this profile, please refer to Dhanya and Hasan (2021).

}, issn = {2666-1667}, doi = {10.1016/j.xpro.2021.101105}, author = {Dhanya, Sreeja Kumari and Hasan, Gaiti} } @article {105156, title = {Deficits Associated With Loss of STIM1 in Purkinje Neurons Including Motor Coordination Can Be Rescued by Loss of Septin 7.}, journal = {Front Cell Dev Biol}, volume = {9}, year = {2021}, month = {2021}, pages = {794807}, abstract = {

Septins are cytoskeletal proteins that can assemble to form heteromeric filamentous complexes and regulate a range of membrane-associated cellular functions. SEPT7, a member of the septin family, functions as a negative regulator of the plasma membrane-localized store-operated Ca entry (SOCE) channel, Orai in neurons, and in human neural progenitor cells. Knockdown of STIM, a Ca sensor in the endoplasmic reticulum (ER) and an integral component of SOCE, leads to flight deficits in that can be rescued by partial loss of SEPT7 in neurons. Here, we tested the effect of reducing and removing SEPT7 in mouse Purkinje neurons (PNs) with the loss of STIM1. Mice with the complete knockout of STIM1 in PNs exhibit several age-dependent changes. These include altered gene expression in PNs, which correlates with increased synapses between climbing fiber (CF) axons and Purkinje neuron (PN) dendrites and a reduced ability to learn a motor coordination task. Removal of either one or two copies of the gene in PNs restored the expression of a subset of genes, including several in the category of neuron projection development. Importantly, the rescue of gene expression in these animals is accompanied by normal CF-PN innervation and an improved ability to learn a motor coordination task in aging mice. Thus, the loss of SEPT7 in PNs further modulates cerebellar circuit function in animals. Our findings are relevant in the context of identifying SEPT7 as a putative therapeutic target for various neurodegenerative diseases caused by reduced intracellular Ca signaling.

}, issn = {2296-634X}, doi = {10.3389/fcell.2021.794807}, author = {Dhanya, Sreeja Kumari and Hasan, Gaiti} } @article {94543, title = {IP3-mediated Ca2+ signals regulate larval to pupal transition under nutrient stress through the H3K36 methyltransferase Set2.}, journal = {Development}, volume = {148}, year = {2021}, month = {2021 Jun 01}, abstract = {

Persistent loss of dietary protein usually signals a shutdown of key metabolic pathways. In Drosophila larvae that have reached a {\textquoteright}critical weight{\textquoteright} and can pupariate to form viable adults, such a metabolic shutdown would needlessly lead to death. Inositol 1,4,5-trisphosphate-mediated calcium (IP3/Ca2+) release in some interneurons (vGlutVGN6341) allows Drosophila larvae to pupariate on a protein-deficient diet by partially circumventing this shutdown through upregulation of neuropeptide signaling and the expression of ecdysone synthesis genes. Here, we show that IP3/Ca2+ signals in vGlutVGN6341 neurons drive expression of Set2, a gene encoding Drosophila Histone 3 Lysine 36 methyltransferase. Furthermore, Set2 expression is required for larvae to pupariate in the absence of dietary protein. IP3/Ca2+ signal-driven Set2 expression upregulates key Ca2+-signaling genes through a novel positive-feedback loop. Transcriptomic studies, coupled with analysis of existing ChIP-seq datasets, identified genes from larval and pupal stages that normally exhibit robust H3K36 trimethyl marks on their gene bodies and concomitantly undergo stronger downregulation by knockdown of either the intracellular Ca2+ release channel IP3R or Set2. IP3/Ca2+ signals thus regulate gene expression through Set2-mediated H3K36 marks on select neuronal genes for the larval to pupal transition.

}, issn = {1477-9129}, doi = {10.1242/dev.199018}, author = {Mitra, Rishav and Richhariya, Shlesha and Jayakumar, Siddharth and Notani, Dimple and Hasan, Gaiti} } @article {92431, title = {Purkinje neurons with loss of STIM1 exhibit age-dependent changes in gene expression and synaptic components}, journal = {J Neurosci.}, year = {2021}, month = {03/2021}, abstract = {

The Stromal interaction molecule 1 (STIM1), is an ER-Ca2+ sensor and an essential component of ER-Ca2+ store operated Ca2+ entry (SOCE). Loss of STIM1 affects metabotropic Glutamate Receptor 1 (mGluR1) mediated synaptic transmission, neuronal Ca2+ homeostasis and intrinsic plasticity in Purkinje Neurons (PNs). Long-term changes of intracellular Ca2+ signaling in PNs lead to neurodegenerative conditions, as evident in individuals with mutations of the ER-Ca2+ channel, the Inositol 1,4,5-triphosphate receptor (IP3R). Here, we asked if changes in such intrinsic neuronal properties, due to loss of STIM1, have an age-dependent impact on PNs. Consequently, we analyzed mRNA expression profiles and cerebellar morphology in PN specific STIM1 knockout mice (STIM1PKO ) of both sexes across ages. Our study identified a requirement for STIM1 mediated Ca2+ signaling in maintaining the expression of genes belonging to key biological networks of synaptic function and neurite development amongst others. Gene expression changes correlated with altered patterns of dendritic morphology and greater innervation of PN dendrites by climbing fibers, in ageing STIM1PKO mice. Together, our data identify STIM1 as an important regulator of Ca2+ homeostasis and neuronal excitability in turn required for maintaining the optimal transcriptional profile of PNs with age. Our findings are significant in the context of understanding how dysregulated calcium signals impact cellular mechanisms in multiple neurodegenerative disorders.Significance StatementIn Purkinje neurons (PNs) the Stromal interaction molecule 1 (STIM1) is required for mGluR1-dependent synaptic transmission, refilling of ER Ca2+ stores, regulation of spike frequency and cerebellar memory consolidation. Here, we provide evidence for a novel role of STIM1 in maintaining the gene expression profile and optimal synaptic connectivity of PNs. Expression of genes related to neurite development and synaptic organization networks are altered in PNs with persistent loss of STIM1. In agreement with these findings the dendritic morphology of PNs and climbing fiber innervations on PNs also undergo significant changes with age. These findings identify a new role for dysregulated intracellular calcium signaling in neurodegenerative disorders and provide novel therapeutic insights.

}, doi = {10.1523/JNEUROSCI.2401-20.2021}, author = {Dhanya, SK and Hasan, Gaiti} } @article {84948, title = {Modulation of flight and feeding behaviours requires presynaptic IPRs in dopaminergic neurons.}, journal = {Elife}, volume = {9}, year = {2020}, month = {2020 Nov 06}, abstract = {

Innate behaviours, although robust and hard wired, rely on modulation of neuronal circuits, for eliciting an appropriate response according to internal states and external cues. flight is one such innate behaviour that is modulated by intracellular calcium release through inositol 1,4,5-trisphosphate receptors (IPRs). Cellular mechanism(s) by which IPRs modulate neuronal function for specific behaviours remain speculative, in vertebrates and invertebrates. To address this, we generated an inducible dominant negative form of the IPR (IPR). Flies with neuronal expression of IPR exhibit flight deficits. Expression of IPR helped identify key flight-modulating dopaminergic neurons with axonal projections in the mushroom body. Flies with attenuated IPRs in these presynaptic dopaminergic neurons exhibit shortened flight bouts and a disinterest in seeking food, accompanied by reduced excitability and dopamine release upon cholinergic stimulation. Our findings suggest that the same neural circuit modulates the drive for food search and for undertaking longer flight bouts.

}, issn = {2050-084X}, doi = {10.7554/eLife.62297}, author = {Sharma, Anamika and Hasan, Gaiti} } @article {83650, title = {Regulation of neuronal physiology by Ca2+ release through the IP3R}, journal = {Current Opinion in Physiology}, volume = {17}, year = {2020}, month = {10/10}, pages = {1-8}, abstract = {

Identification of mutations in the gene encoding the inositol 1,4,5-trisphosphate receptor Type 1 (IP3R1) as causative for Spinocerebellar Ataxia 15, 29 and Gillespie Syndrome emphasize the importance of understanding how the IP3R impacts neuronal function and physiology. Here we discuss how cellular changes due to IP3 mediated Ca2+release affect systemic physiology in Drosophila and mouse. The relevance of these findings to recently identified human neurological conditions are also discussed.

}, doi = {10.1016/j.cophys.2020.06.001}, author = {Hasan, Gaiti and Sharma, Anamika} } @article {79802, title = {SEPT7 regulates Ca entry through Orai channels in human neural progenitor cells and neurons.}, journal = {Cell Calcium}, volume = {90}, year = {2020}, month = {2020 Jul 09}, pages = {102252}, abstract = {

Human neural progenitor cells (hNPCs) are self-renewing cells of neural lineage that can be differentiated into neurons of different subtypes. Here we show that SEPT7, a member of the family of filament-forming GTPases called septins, prevents constitutive Ca entry through the store-operated Ca entry channel, Orai in hNPCs and in differentiated neurons and is thus required for neuronal calcium homeostasis. Previous work in Drosophila neurons has shown that loss of one copy of the evolutionarily-conserved dSEPT7 gene leads to elevated Ca entry via Orai, in the absence of ER-Ca store depletion. We have identified an N-terminal polybasic region of SEPT7, known to interact with membrane-localized phospholipids, as essential for spontaneous calcium entry through Orai in hNPCs, whereas the GTPase domain of dSEPT7 is dispensable for this purpose. Re-organisation of Orai1 and the ER-Ca sensor STIM1 observed near the plasma membrane in SEPT7 KD hNPCs, supports the idea that Septin7 containing heteromers prevent Ca entry through a fraction of STIM-Orai complexes. Possible mechanisms by which SEPT7 reduction leads to opening of Orai channels in the absence of store-depletion are discussed.

}, issn = {1532-1991}, doi = {10.1016/j.ceca.2020.102252}, author = {Deb, Bipan K and Chakraborty, Pragnya and Gopurappilly, Renjitha and Hasan, Gaiti} } @article {79797, title = {Surviving nutritional deprivation during development: neuronal intracellular calcium signaling is critical.}, journal = {Int J Dev Biol}, volume = {64}, year = {2020}, month = {2020}, pages = {249-256}, abstract = {

Developing cells and tissues in a growing animal need to sense food quality and integrate this information with on-going time-bound developmental programs. The integration of metabolism with development requires cellular and systemic coordination. Work in our laboratory has focused on Ca signaling arising from the release of Ca stored in the endoplasmic reticulum (ER), which triggers store-operated Ca entry. We describe a role for ER-store Ca that operates at the cellular level in various classes of neurons, and eventually drives the systemic coordination required to survive and complete development under conditions of nutritional deprivation. In the model system Drosophila melanogaster, we have developed a paradigm to induce nutritional stress during the larval stage and used pupariation as a read-out for development. Applying the vast genetic tool kit available in Drosophila to this paradigm, we have uncovered novel roles for intracellular Ca signaling in regulating neuronal activity, at the level of transcription in glutamatergic neurons, and translation in neuropeptidergic neurons. We find that such regulation of cellular processes is critical for integrating information across a neural circuit at multiple levels, starting from the point of sensing systemic and environmental levels of amino acids to finally connecting with neuropeptide secreting neurons, that communicate with the prothoracic gland, an organ that makes the key developmental hormone, ecdysone. This work underscores the importance of ER-store Ca for neuronal health, with consequences for animal development.

}, issn = {1696-3547}, doi = {10.1387/ijdb.190148mm}, author = {Hasan, Gaiti} } @article {62763, title = {ER-Ca2+ sensor STIM regulates neuropeptides required for development under nutrient restriction in Drosophila.}, journal = {PLoS One}, volume = {14}, year = {2019}, month = {2019}, pages = {e0219719}, abstract = {

Neuroendocrine cells communicate via neuropeptides to regulate behaviour and physiology. This study examines how STIM (Stromal Interacting Molecule), an ER-Ca2+ sensor required for Store-operated Ca2+ entry, regulates neuropeptides required for Drosophila development under nutrient restriction (NR). We find two STIM-regulated peptides, Corazonin and short Neuropeptide F, to be required for NR larvae to complete development. Further, a set of secretory DLP (Dorso lateral peptidergic) neurons which co-express both peptides was identified. Partial loss of dSTIM caused peptide accumulation in the DLPs, and reduced systemic Corazonin signalling. Upon NR, larval development correlated with increased peptide levels in the DLPs, which failed to occur when dSTIM was reduced. Comparison of systemic and cellular phenotypes associated with reduced dSTIM, with other cellular perturbations, along with genetic rescue experiments, suggested that dSTIM primarily compromises neuroendocrine function by interfering with neuropeptide release. Under chronic stimulation, dSTIM also appears to regulate neuropeptide synthesis.

}, issn = {1932-6203}, doi = {10.1371/journal.pone.0219719}, author = {Wegener, Christian and Hasan, Gaiti} } @article {51001, title = {Extended Flight Bouts Require Disinhibition from GABAergic Mushroom Body Neurons.}, journal = {Curr Biol}, volume = {29}, year = {2019}, month = {2019 Jan 21}, pages = {283-293.e5}, abstract = {

Insect flight is a complex behavior that requires the integration of multiple sensory inputs with flight motor output. Although previous genetic studies identified central brain monoaminergic neurons that modulate Drosophila flight, neuro-modulatory circuits underlying sustained flight bouts remain unexplored. Certain classes of dopaminergic and octopaminergic neurons that project to the mushroom body, a higher integrating center in the insect brain, are known to modify neuronal output based on contextual cues and thereby organismal behavior. This study focuses on how monoaminergic modulation of mushroom body GABAergic output neurons (MBONs) regulates the duration of flight bouts. Octopaminergic neurons in the sub-esophageal zone stimulate central dopaminergic neurons (protocerebral anterior medial, PAM) that project to GABAergic MBONs. Either inhibition of octopaminergic and dopaminergic neurons or activation of GABAergic MBONs reduces the duration of flight bouts. Moreover, activity in the PAM neurons inhibits the GABAergic MBONs. Our data suggest that disinhibition of the identified neural circuit very likely occurs after flight initiation and is required to maintain the "flight state" when searching for distant sites, possibly related to food sources, mating partners, or a suitable egg-laying site. VIDEO ABSTRACT.

}, issn = {1879-0445}, doi = {10.1016/j.cub.2018.11.070}, author = {Manjila, Steffy B and Kuruvilla, Maria and Ferveur, Jean-Fran{\c c}ois and Sane, Sanjay P and Hasan, Gaiti} } @article {59728, title = {Formal structures of mentorship in universities and research institutions will benefit both science and scientists}, journal = {Current Science}, volume = {116}, year = {2019}, month = {05/2019}, pages = {1615-1616}, url = {https://www.currentscience.ac.in/Volumes/116/10/1615.pdf}, author = {Hasan, Gaiti} } @article {64328, title = {Implications of the null mutation for synapsin phosphorylation, longevity, climbing, and behavioural plasticity in adult .}, journal = {J Exp Biol}, year = {2019}, month = {2019 Sep 05}, abstract = {

The gene of encodes a highly abundant 47 kDa synaptic vesicle-associated protein. null mutants show defects in synaptic plasticity and larval olfactory associative learning but the molecular function of Sap47 at the synapse is unknown. We demonstrate that Sap47 modulates the phosphorylation of another highly abundant conserved presynaptic protein, synapsin. Site-specific phosphorylation of synapsin has repeatedly been shown to be important for behavioural plasticity but information of where in the brain phospho-synapsin isoforms are localized has been lacking. Here we report the distribution of serine-6-phosphorylated synapsin in the adult brain and show that it is highly enriched in rings of synapses in the ellipsoid body, and in large synapses near the lateral triangle. Effects of knock-out of or on olfactory associative learning/memory are compatible with the hypothesis that both proteins operate in the same molecular pathway. We therefore asked if this might also hold for other aspects of their function. We show that knock-out of but not reduces life span, whereas knock-out of , or both affects climbing as well as plasticity in circadian rhythms and sleep. Furthermore, electrophysiological assessment of synaptic properties at the larval neuromuscular junction (NMJ) reveals increased spontaneous synaptic vesicle fusion and reduced paired pulse facilitation in and single and double mutants. Our results imply that Sap47 and synapsin cooperate non-uniformly in the control of synaptic properties in different behaviourally relevant neuronal networks of the fruitfly.

}, issn = {1477-9145}, doi = {10.1242/jeb.203505}, author = {Blanco-Redondo, Beatriz and Nuwal, Nidhi and Kneitz, Susanne and Nuwal, Tulip and Halder, Partho and Liu, Yiting and Ehmann, Nadine and Scholz, Nicole and Mayer, Annika and Kleber, J{\"o}rg and K{\"a}hne, Thilo and Schmitt, Dominique and Sadanandappa, Madhumala K and Funk, Natalja and Albertova, Viera and Helfrich-F{\"o}rster, Charlotte and Ramaswami, Mani and Hasan, Gaiti and Kittel, Robert J and Langenhan, Tobias and Gerber, Bertram and Buchner, Erich} } @article {47058, title = {IP3 receptors and Ca2+ entry}, journal = {Biochim Biophys Acta Mol Cell Res}, volume = {1866}, year = {2019}, month = {2018 Nov 15}, pages = {1092-1100}, abstract = {

Inositol 1,4,5-trisphosphate receptors (IPR) are the most widely expressed intracellular Ca release channels. Their activation by IP and Ca allows Ca to pass rapidly from the ER lumen to the cytosol. The resulting increase in cytosolic [Ca] may directly regulate cytosolic effectors or fuel Ca uptake by other organelles, while the decrease in ER luminal [Ca] stimulates store-operated Ca entry (SOCE). We are close to understanding the structural basis of both IPR activation, and the interactions between the ER Ca-sensor, STIM, and the plasma membrane Ca channel, Orai, that lead to SOCE. IPRs are the usual means through which extracellular stimuli, through ER Ca release, stimulate SOCE. Here, we review evidence that the IPRs most likely to respond to IP are optimally placed to allow regulation of SOCE. We also consider evidence that IPRs may regulate SOCE downstream of their ability to deplete ER Ca stores. Finally, we review evidence that IPRs in the plasma membrane can also directly mediate Ca entry in some cells.

}, issn = {1879-2596}, doi = {10.1016/j.bbamcr.2018.11.007}, author = {Thillaiappan, Nagendra Babu and Chakraborty, Pragnya and Hasan, Gaiti and Taylor, Colin W} } @article {62768, title = {Measurement of Store-Operated Calcium Entry in Human Neural Cells: From Precursors to Differentiated Neurons.}, journal = {Methods Mol Biol}, volume = {2029}, year = {2019}, month = {2019}, pages = {257-271}, abstract = {

Calcium imaging in an ex-vivo\ setup is used to understand the calcium status of isolated cells or tissue. In this chapter we explain the use of the ratiometric chemical indicator Fura-2 which can be loaded into isolated cells in the form of lipophilic acetomethyl (AM) esters. Fura-2 is a combination of calcium chelator and fluorophore, and can be used with dual wavelength excitation (340/380\ nm) for quantitative calcium concentrations. The cells can then be viewed using a fluorescence microscope and captured by a CCD camera. We specifically discuss the technique involved in understanding the endoplasmic reticulum (ER)-driven store-operated calcium entry (SOCE) in human neural precursors (NPCs)\ and spontaneously differentiated neurons derived from a pluripotent human\ embryonic stem cell\ (hESC) line. The derivation of neural precursors from stem cells and their subsequent spontaneous neural differentiation is also explained. The method can be used for various non-excitable and excitable cell types including neurons, be it freshly isolated, from frozen vials, or derived from different stem cell lines.

}, issn = {1940-6029}, doi = {10.1007/978-1-4939-9631-5_20}, author = {Gopurappilly, Renjitha and Deb, Bipan Kumar and Chakraborty, Pragnya and Hasan, Gaiti} } @article {36657, title = {dSTIM- and Ral/Exocyst-Mediated Synaptic Release from Pupal Dopaminergic Neurons Sustains Flight.}, journal = {eNeuro}, volume = {5}, year = {2018}, month = {2018 May-Jun}, abstract = {

Manifestation of appropriate behavior in adult animals requires developmental mechanisms that help in the formation of correctly wired neural circuits. Flight circuit development in requires store-operated calcium entry (SOCE) through the STIM/Orai pathway. SOCE-associated flight deficits in adult derive extensively from regulation of gene expression in pupal neurons, and one such SOCE-regulated gene encodes the small GTPase . The cellular mechanism by which Ral helps in maturation of the flight circuit was not understood. Here, we show that knockdown of components of a Ral effector, the exocyst complex, in pupal neurons also leads to reduced flight bout durations, and this phenotype derives primarily from dopaminergic neurons. Importantly, synaptic release from pupal dopaminergic neurons is abrogated upon knockdown of dSTIM, Ral, or exocyst components. overexpression restores the diminished synaptic release of knockdown neurons as well as flight deficits associated with knockdown in dopaminergic neurons. These results identify Ral-mediated vesicular release as an effector mechanism of neuronal SOCE in pupal dopaminergic neurons with functional consequences on flight behavior.

}, issn = {2373-2822}, doi = {10.1523/ENEURO.0455-17.2018}, author = {Richhariya, Shlesha and Jayakumar, Siddharth and Kumar Sukumar, Sanjay and Hasan, Gaiti} } @article {43850, title = {FMRFa receptor stimulated Ca2+ signals alter the activity of flight modulating central dopaminergic neurons in Drosophila melanogaster.}, journal = {PLoS Genet}, volume = {14}, year = {2018}, month = {2018 Aug}, pages = {e1007459}, abstract = {

Neuropeptide signaling influences animal behavior by modulating neuronal activity and thus altering circuit dynamics. Insect flight is a key innate behavior that very likely requires robust neuromodulation. Cellular and molecular components that help modulate flight behavior are therefore of interest and require investigation. In a genetic RNAi screen for G-protein coupled receptors that regulate flight bout durations, we earlier identified several receptors, including the receptor for the neuropeptide FMRFa (FMRFaR). To further investigate modulation of insect flight by FMRFa we generated CRISPR-Cas9 mutants in the gene encoding the Drosophila FMRFaR. The mutants exhibit significant flight deficits with a focus in dopaminergic cells. Expression of a receptor specific RNAi in adult central dopaminergic neurons resulted in progressive loss of sustained flight. Further, genetic and cellular assays demonstrated that FMRFaR stimulates intracellular calcium signaling through the IP3R and helps maintain neuronal excitability in a subset of dopaminergic neurons for positive modulation of flight bout durations.

}, issn = {1553-7404}, doi = {10.1371/journal.pgen.1007459}, author = {Ravi, Preethi and Trivedi, Deepti and Hasan, Gaiti} } @article {45308, title = {A multi-component neuronal response encodes the larval decision to pupariate upon amino acid starvation.}, journal = {J Neurosci}, year = {2018}, month = {2018 Oct 09}, abstract = {

Organisms need to co-ordinate growth with development, particularly in the context of nutrient availability. Thus, multiple ways have evolved to survive extrinsic nutrient deprivation during development. In , growth occurs during larval development. Larvae are thus critically dependant on nutritional inputs but post critical weight they pupariate even when starved. How nutrient availability is coupled to the internal metabolic state for the decision to pupariate needs better understanding. We had earlier identified glutamatergic interneurons in the ventral ganglion that regulate pupariation on a protein-deficient diet. Here we report that third instar larvae (either sex) sense arginine to evaluate their nutrient environment using an amino-acid transporter Slimfast. The glutamatergic interneurons integrate external protein availability with internal metabolic state through neuropeptide signals. IP mediated calcium release and store-operated calcium entry are essential in these glutamatergic neurons for such integration and alter neuronal function by reducing the expression of multiple ion channels.Co-ordinating growth with development, in the context of nutrient availability is a challenge for all organisms in nature. Post attainment of "critical weight" insect larvae can pupariate even in the absence of nutrition. Mechanism(s) that stimulate appropriate cellular responses and allow normal development on a nutritionally deficient diet remain to be understood. Here, we demonstrate that nutritional deprivation, in post-critical weight larvae, is sensed by special sensory neurons through an amino acid transporter that detects loss of environmental arginine. This information is integrated by glutamatergic interneurons with the internal metabolic state through neuropeptide signals. These glutamatergic interneurons require calcium-signalling regulated expression of a host of neuronal channels to generate complex calcium signals essential for pupariation on a protein-deficient diet.

}, issn = {1529-2401}, doi = {10.1523/JNEUROSCI.1163-18.2018}, author = {Jayakumar, Siddharth and Richhariya, Shlesha and Deb, Bipan Kumar and Hasan, Gaiti} } @article {33716, title = {Neuronal Calcium Signaling in Metabolic Regulation and Adaptation to Nutrient Stress.}, journal = {Front Neural Circuits}, volume = {12}, year = {2018}, month = {2018}, pages = {25}, abstract = {

All organisms can respond physiologically and behaviorally to environmental fluxes in nutrient levels. Different nutrient sensing pathways exist for specific metabolites, and their inputs ultimately define appropriate nutrient uptake and metabolic homeostasis. Nutrient sensing mechanisms at the cellular level require pathways such as insulin and target of rapamycin (TOR) signaling that integrates information from different organ systems like the fat body and the gut. Such integration is essential for coordinating growth with development. Here we review the role of a newly identified set of integrative interneurons and the role of intracellular calcium signaling within these neurons, in regulating nutrient sensing under conditions of nutrient stress. A comparison of the identified circuit and cellular mechanisms employed in this circuit, with vertebrate systems, suggests that the identified cell signaling mechanisms may be conserved for neural circuit function related to nutrient sensing by central neurons. The ideas proposed are potentially relevant for understanding the molecular basis of metabolic disorders, because these are frequently linked to nutritional stress.

}, issn = {1662-5110}, doi = {10.3389/fncir.2018.00025}, author = {Jayakumar, Siddharth and Hasan, Gaiti} } @article {39425, title = {SEPT7-mediated regulation of Ca entry through Orai channels requires other septin subunits.}, journal = {Cytoskeleton (Hoboken)}, volume = {76}, year = {2018}, month = {2018 Jul 13}, pages = {104-114}, abstract = {

Orai channels are plasma membrane resident Ca channels that allow extracellular Ca uptake after depletion of ER-Ca stores by a process called store-operated Ca entry (SOCE). Septins of the SEPT2 subgroup act as positive regulators of SOCE in human non-excitable cells. SEPT2 subgroup septins form the central core of hetero-hexameric or hetero-octameric complexes with SEPT6, SEPT7 and SEPT9 subgroup septins. The presence of fewer septin encoding genes coupled with ease of genetic manipulation allows for better understanding of septin subgroup function in Drosophila. Our earlier findings show that although dSEPT7 reduction does not alter Orai-mediated Ca entry during SOCE, it results in constitutive activation of Orai channels in resting neurons. Here we have investigated the role of other septin subgroup members in regulating Orai channel activation in Drosophila neurons by both cellular and functional assays. We show that dSEPT1, a SEPT2 subgroup septin can exist in a complex with dSEPT2 and dSEPT7 in the central nervous system (CNS) of Drosophila. Our findings suggest that the nature of septin filaments and heteromers obtained after reducing septins of different subgroups alters their ability to regulate Orai channel opening. The molecular mechanisms underlying this complex regulation of Orai function by septins require further cellular investigations. This article is protected by copyright. All rights reserved.

}, issn = {1949-3592}, doi = {10.1002/cm.21476}, author = {Deb, Bipan Kumar and Hasan, Gaiti} } @article {36788, title = {Stable Knockdown in Self-Renewing Human Neural Precursors Promotes Premature Neural Differentiation.}, journal = {Front Mol Neurosci}, volume = {11}, year = {2018}, month = {2018}, pages = {178}, abstract = {

Ca signaling plays a significant role in the development of the vertebrate nervous system where it regulates neurite growth as well as synapse and neurotransmitter specification. Elucidating the role of Ca signaling in mammalian neuronal development has been largely restricted to either small animal models or primary cultures. Here we derived human neural precursor cells (NPCs) from human embryonic stem cells to understand the functional significance of a less understood arm of calcium signaling, Store-operated Ca entry or SOCE, in neuronal development. Human NPCs exhibited robust SOCE, which was significantly attenuated by expression of a stable shRNA-miR targeted toward the SOCE molecule, STIM1. Along with the plasma membrane channel Orai, STIM is an essential component of SOCE in many cell types, where it regulates gene expression. Therefore, we measured global gene expression in human NPCs with and without knockdown. Interestingly, pathways down-regulated through knockdown were related to cell proliferation and DNA replication processes, whereas post-synaptic signaling was identified as an up-regulated process. To understand the functional significance of these gene expression changes we measured the self-renewal capacity of NPCs with knockdown. The knockdown NPCs demonstrated significantly reduced neurosphere size and number as well as precocious spontaneous differentiation toward the neuronal lineage, as compared to control cells. These findings demonstrate that mediated SOCE in human NPCs regulates gene expression changes, that are likely to physiologically modulate the self-renewal and differentiation of NPCs.

}, issn = {1662-5099}, doi = {10.3389/fnmol.2018.00178}, author = {Gopurappilly, Renjitha and Deb, Bipan Kumar and Chakraborty, Pragnya and Hasan, Gaiti} } @article {43844, title = {Store-Operated Ca Entry in Drosophila Primary Neuronal Cultures.}, journal = {Methods Mol Biol}, volume = {1843}, year = {2018}, month = {2018}, pages = {125-136}, abstract = {

Intracellular calcium signals in neurons frequently derive from the stimulation of G protein-coupled receptors (GPCR) by neurotransmitters, neuropeptides, and neurohormones. GPCR stimulation in neurons leads to generation of inositol 1,4,5-trisphosphate (IP), which in turn activates endoplasmic reticulum (ER)-localized IP receptors. The IP receptor\ (IPR) is a ligand-gated Ca channel, which releases Ca from ER stores. In Drosophila neurons it has been shown that depletion of ER Ca store is followed by store-operated Ca entry (SOCE) through STIM\ and Orai, the ER Ca sensor and the plasma membrane Ca channel respectively. The elucidation of this Ca signaling pathway in neurons has in part been possible\ due to the ease of genetic manipulation in Drosophila, which has allowed neuron-specific knockdown of various proteins of interest. This has been followed by standardization of conditions for culturing neurons from dissected brains of the relevant genotypes, such that they could be used for robust Ca measurements by imaging with standard Ca indicator dyes. Protocols for measurement of IP-mediated Ca release, passive depletion of ER Ca store, and SOCE in primary cultures of Drosophila neurons are described here.

}, issn = {1940-6029}, doi = {10.1007/978-1-4939-8704-7_11}, author = {Chakraborty, Sumita and Hasan, Gaiti} } @article {24297, title = {Control of protein translation by IP3R-mediated Ca(2+) release in Drosophila neuroendocrine cells.}, journal = {Fly (Austin)}, year = {2017}, month = {2017 Sep 26}, abstract = {

The inositol 1,4,5-trisphosphate receptor (IP3R) is one of two Ca(2+) channels that gates Ca(2+) release from ER-stores. The ligand IP3, generated upon specific G-protein coupled receptor activation, binds to IP3R to release Ca(2+) into the cytosol. IP3R also mediates ER-store Ca(2+) release into the mitochondria, under basal as well as stimulatory conditions; an activity that influences cellular bioenergetics and thus, cellular growth and proliferation. In Drosophila neuroendocrine cells expressing a hypomorphic mutant of IP3R, we observed reduced protein translation levels. Here, we discuss the possible molecular mechanism for this observation. We hypothesise that the cellular energy sensor, AMPK connects IP3R mediated Ca(2+) release into the mitochondria, to protein translation, via the TOR pathway.

}, issn = {1933-6942}, doi = {10.1080/19336934.2017.1384103}, author = {Hasan, Gaiti} } @article {17522, title = {CRISPR-Cas-Induced Mutants Identify a Requirement for dSTIM in Larval Dopaminergic Cells of Drosophila melanogaster.}, journal = {G3 (Bethesda)}, volume = {7}, year = {2017}, month = {2017 Mar 10}, pages = {923-933}, abstract = {

Molecular components of store-operated calcium entry have been identified in the recent past and consist of the endoplasmic reticulum (ER) membrane-resident calcium sensor STIM and the plasma membrane-localized calcium channel Orai. The physiological function of STIM and Orai is best defined in vertebrate immune cells. However, genetic studies with RNAi strains in Drosophila suggest a role in neuronal development and function. We generated a CRISPR-Cas-mediated deletion for the gene encoding STIM in Drosophila (dSTIM), which we demonstrate is larval lethal. To study STIM function in neurons, we merged the CRISPR-Cas9 method with the UAS-GAL4 system to generate either tissue- or cell type-specific inducible STIM knockouts (KOs). Our data identify an essential role for STIM in larval dopaminergic cells. The molecular basis for this cell-specific requirement needs further investigation.

}, issn = {2160-1836}, doi = {10.1534/g3.116.038539}, author = {Pathak, Trayambak and Trivedi, Deepti and Hasan, Gaiti} } @article {14685, title = {IP3R mediated Ca(2+) release regulates protein metabolism in Drosophila neuroendocrine cells: implications for development under nutrient stress.}, journal = {Development}, year = {2017}, month = {2017 Mar 13}, abstract = {

Successful completion of animal development is fundamentally reliant on nutritional cues. Adaptations for surviving nutritional loss are coordinated in part by neural circuits. As neuropeptides secreted by neuroendocrine (NE) cells critically modulate neural circuits, we investigated NE cell function during development under nutrient stress. Starved Drosophila larvae exhibited reduced pupariation, if either insulin signaling or IP3/Ca(2+) signaling, were down-regulated in NE cells. Moreover, an IP3R (Inositol 1,4,5-trisphosphate receptor) loss-of-function mutant displayed reduced protein synthesis, which was rescued by over-expression of either InR (insulin receptor) or IP3R in NE cells of the mutant, suggesting that the two signaling pathways may be functionally compensatory. Furthermore, cultured IP3R mutant NE cells, but not neurons, exhibited reduced protein translation. Thus cell-specific regulation of protein synthesis by IP3R in NE cells influences protein metabolism. We propose that this regulation helps developing animals survive poor nutritional conditions.

}, issn = {1477-9129}, doi = {10.1242/dev.145235}, author = {Megha, Megha and Hasan, Gaiti} } @article {13641, title = {A pupal transcriptomic screen identifies Ral as a target of store-operated calcium entry in Drosophila neurons.}, journal = {Sci Rep}, volume = {7}, year = {2017}, month = {2017 Feb 14}, pages = {42586}, abstract = {

Transcriptional regulation by Store-operated Calcium Entry (SOCE) is well studied in non-excitable cells. However, the role of SOCE has been poorly documented in neuronal cells with more complicated calcium dynamics. Previous reports demonstrated a requirement for SOCE in neurons that regulate Drosophila flight bouts. We refine this requirement temporally to the early pupal stage and use RNA-sequencing to identify SOCE mediated gene expression changes in the developing Drosophila pupal nervous system. Down regulation of dStim, the endoplasmic reticular calcium sensor and a principal component of SOCE in the nervous system, altered the expression of 131 genes including Ral, a small GTPase. Disruption of Ral function in neurons impaired flight, whereas ectopic expression of Ral in SOCE-compromised neurons restored flight. Through live imaging of calcium transients from cultured pupal neurons, we confirmed that Ral does not participate in SOCE, but acts downstream of it. These results identify neuronal SOCE as a mechanism that regulates expression of specific genes during development of the pupal nervous system and emphasizes the relevance of SOCE-regulated gene expression to flight circuit maturation.

}, issn = {2045-2322}, doi = {10.1038/srep42586}, author = {Richhariya, Shlesha and Jayakumar, Siddharth and Abruzzi, Katharine and Rosbash, Michael and Hasan, Gaiti} } @article {28067, title = {Ral function in muscle is required for flight maintenance in Drosophila.}, journal = {Small GTPases}, year = {2017}, month = {2017 Dec 28}, pages = {1-6}, abstract = {

Ral is a small GTPase of the Ras superfamily that is important for a number of cellular functions. Recently, we found that expression of Ral is regulated by store-operated calcium entry (SOCE) in Drosophila neurons. In this study, through genetic and behavioural experiments, we show that Ral function is required in differentiated muscles for flight. Reducing Ral function in muscles, specifically reduced duration of flight bouts but not other motor functions, like climbing. Interestingly, unlike in the nervous system, Ral expression in the muscle is not regulated by SOCE. Moreover, either knockdown or genetic inhibition of SOCE in muscles does not affect flight. These findings demonstrate that a multiplicity of signalling mechanisms very likely regulate Ral expression in different tissues.

}, issn = {2154-1256}, doi = {10.1080/21541248.2017.1367456}, author = {Richhariya, Shlesha and Hasan, Gaiti} } @article {17148, title = {Spontaneous Ca(2+) Influx in Drosophila Pupal Neurons Is Modulated by IP3-Receptor Function and Influences Maturation of the Flight Circuit.}, journal = {Front Mol Neurosci}, volume = {10}, year = {2017}, month = {2017}, pages = {111}, abstract = {

Inositol 1,4,5-trisphosphate receptors (IP3R) are Ca(2+) channels on the neuronal endoplasmic reticulum (ER) membrane. They are gated by IP3, produced upon external stimulation and activation of G protein-coupled receptors on the plasma membrane (PM). IP3-mediated Ca(2+) release, and the resulting depletion of the ER store, triggers entry of extracellular Ca(2+) by store-operated Ca(2+) entry (SOCE). Mutations in IP3R attenuate SOCE. Compromised IP3R function and SOCE during pupal development of Drosophila leads to flight deficits and mimics suppression of neuronal activity during pupal or adult development. To understand the effect of compromised IP3R function on pupal neuronal calcium signaling, we examined the effects of mutations in the IP3R gene (itpr) on Ca(2+) signals in cultured neurons derived from Drosophila pupae. We observed increased spontaneous Ca(2+) influx across the PM of isolated pupal neurons with mutant IP3R and also a loss of SOCE. Both spontaneous Ca(2+) influx and reduced SOCE were reversed by over-expression of dOrai and dSTIM, which encode the SOCE Ca(2+) channel and the ER Ca(2+)-sensor that regulates it, respectively. Expression of voltage-gated Ca(2+) channels (cac, Ca-α1D and Ca-αT) was significantly reduced in itpr mutant neurons. However, expression of trp mRNAs and transient receptor potential (TRP) protein were increased, suggesting that TRP channels might contribute to the increased spontaneous Ca(2+) influx in neurons with mutant IP3R. Thus, IP3R/SOCE modulates spontaneous Ca(2+) influx and expression of PM Ca(2+) channels in Drosophila pupal neurons. Spontaneous Ca(2+) influx compensates for the loss of SOCE in Drosophilaitpr mutant neurons.

}, doi = {10.3389/fnmol.2017.00111}, author = {Chakraborty, Sumita and Hasan, Gaiti} } @article {7042, title = {Drosophila larval to pupal switch under nutrient stress requires IP3R/Ca(2+) signalling in glutamatergic interneurons.}, journal = {Elife}, volume = {5}, year = {2016}, month = {2016 Aug 5}, abstract = {

Neuronal circuits are known to integrate nutritional information, but the identity of the circuit components is not completely understood. Amino acids are a class of nutrients that are vital for the growth and function of an organism. Here, we report a neuronal circuit that allows Drosophila larvae to overcome amino acid deprivation and pupariate. We find that nutrient stress is sensed by the class IV multidendritic cholinergic neurons. Through live calcium imaging experiments, we show that these cholinergic stimuli are conveyed to glutamatergic neurons in the ventral ganglion through mAChR. We further show that IP3R-dependent calcium transients in the glutamatergic neurons convey this signal to downstream medial neurosecretory cells (mNSCs). The circuit ultimately converges at the ring gland and regulates expression of ecdysteroid biosynthetic genes. Activity in this circuit is thus likely to be an adaptation that provides a layer of regulation to help surpass nutritional stress during development.

}, issn = {2050-084X}, doi = {10.7554/eLife.17495}, author = {Jayakumar, Siddharth and Richhariya, Shlesha and Reddy, O Venkateswara and Texada, Michael J and Hasan, Gaiti} } @article {7589, title = {Mutant IP3 receptors attenuate store-operated Ca2+ entry by destabilizing STIM-Orai interactions in Drosophila neurons.}, journal = {J Cell Sci}, year = {2016}, month = {2016 Sep 2}, abstract = {

Store-operated Ca(2+) entry (SOCE) occurs when loss of Ca(2+) from the endoplasmic reticulum (ER) stimulates the Ca(2+) sensor, STIM, to cluster and activate the plasma membrane (PM) Ca(2+) channel, Orai. Inositol 1,4,5-trisphosphate receptors (IP3R) are assumed to regulate SOCE solely by mediating ER Ca(2+) release. We show that in Drosophila neurons, mutant IP3R attenuate SOCE evoked by depleting Ca(2+) stores with thapsigargin. In normal neurons, store depletion caused STIM and IP3R to accumulate near the PM, association of STIM with Orai, clustering of STIM and Orai at ER-PM junctions, and activation of SOCE. These responses were attenuated in neurons with mutant IP3R and rescued by over-expression of STIM with Orai. We conclude that after depletion of Ca(2+) stores in Drosophila, translocation of IP3R to ER-PM junctions facilitates the coupling of STIM to Orai that leads to activation of SOCE.

}, issn = {1477-9137}, doi = {10.1242/jcs.191585}, author = {Chakraborty, Sumita and Deb, Bipan K and Chorna, Tetyana and Konieczny, Vera and Taylor, Colin W and Hasan, Gaiti} } @article {11585, title = {Regulation of Store-Operated Ca(2+) Entry by Septins.}, journal = {Front Cell Dev Biol}, volume = {4}, year = {2016}, month = {2016}, pages = {142}, abstract = {

The mechanism of store-operated Ca(2+) entry (SOCE) brings extracellular Ca(2+) into cells after depletion of intracellular Ca(2+) stores. Regulation of Ca(2+) homeostasis by SOCE helps control various intracellular signaling functions in both non-excitable and excitable cells. Whereas essential components of the SOCE pathway are well characterized, molecular mechanisms underlying regulation of this pathway need investigation. A class of proteins recently demonstrated as regulating SOCE is septins. These are filament-forming GTPases that assemble into higher order structures. One of their most studied cellular functions is as a molecular scaffold that creates diffusion barriers in membranes for a variety of cellular processes. Septins regulate SOCE in mammalian non-excitable cells and in Drosophila neurons. However, the molecular mechanism of SOCE-regulation by septins and the contribution of different subgroups of septins to SOCE-regulation remain to be understood. The regulation of SOCE is relevant in multiple cellular contexts as well as in diseases, such as the Severe Combined Immunodeficiency (SCID) syndrome and neurodegenerative syndromes like Alzheimer{\textquoteright}s, Spino-Cerebellar Ataxias and Parkinson{\textquoteright}s. Moreover, Drosophila neurons, where loss of SOCE leads to flight deficits, are a possible cellular template for understanding the molecular basis of neuronal deficits associated with loss of either the Inositol-1,4,5-trisphosphate receptor (IP3R1), a key activator of neuronal SOCE or the Endoplasmic reticulum resident Ca(2+) sensor STIM1 (Stromal Interaction Molecule) in mouse. This perspective summarizes our current understanding of septins as regulators of SOCE and discusses the implications for mammalian neuronal function.

}, doi = {10.3389/fcell.2016.00142}, author = {Deb, Bipan K and Hasan, Gaiti} } @article {3906, title = {Store-independent modulation of Ca(2+) entry through Orai by Septin 7.}, journal = {Nat Commun}, volume = {7}, year = {2016}, month = {2016}, abstract = {

Orai channels are required for store-operated Ca(2+) entry (SOCE) in multiple cell types. Septins are a class of GTP-binding proteins that function as diffusion barriers in cells. Here we show that Septin 7 acts as a {\textquoteright}molecular brake{\textquoteright} on activation of Orai channels in Drosophila neurons. Lowering Septin 7 levels results in dOrai-mediated Ca(2+) entry and higher cytosolic Ca(2+) in resting neurons. This Ca(2+) entry is independent of depletion of endoplasmic reticulum Ca(2+) stores and Ca(2+) release through the inositol-1,4,5-trisphosphate receptor. Importantly, store-independent Ca(2+) entry through Orai compensates for reduced SOCE in the Drosophila flight circuit. Moreover, overexpression of Septin 7 reduces both SOCE and flight duration, supporting its role as a negative regulator of Orai channel function in vivo. Septin 7 levels in neurons can, therefore, alter neural circuit function by modulating Orai function and Ca(2+) homeostasis.

}, issn = {2041-1723}, doi = {10.1038/ncomms11751}, author = {Deb, BipanKumar and Pathak, Trayambak and Hasan, Gaiti} } @article {911, title = {Maturation of a central brain flight circuit in Drosophila requires Fz2/Ca{\texttwosuperior}$^{+}$ signaling.}, journal = {Elife}, volume = {4}, year = {2015}, month = {2015}, abstract = {

The final identity of a differentiated neuron is determined by multiple signaling events, including activity dependent calcium transients. Non-canonical Frizzled2 (Fz2) signaling generates calcium transients that determine neuronal polarity, neuronal migration, and synapse assembly in the developing vertebrate brain. Here, we demonstrate a requirement for Fz2/Ca(2+) signaling in determining the final differentiated state of a set of central brain dopaminergic neurons in Drosophila, referred to as the protocerebral anterior medial (PAM) cluster. Knockdown or inhibition of Fz2/Ca(2+) signaling during maturation of the flight circuit in pupae reduces Tyrosine Hydroxylase (TH) expression in the PAM neurons and affects maintenance of flight. Thus, we demonstrate that Fz2/Ca(2+) transients during development serve as a pre-requisite for normal adult behavior. Our results support a neural mechanism where PAM neuron send projections to the α{\textquoteright} and β{\textquoteright} lobes of a higher brain centre, the mushroom body, and function in dopaminergic re-inforcement of flight.

}, issn = {2050-084X}, doi = {10.7554/eLife.07046}, author = {Agrawal, Tarjani and Hasan, Gaiti} } @article {734, title = {Neural control of wing coordination in flies.}, journal = {Curr Biol}, volume = {25}, year = {2015}, month = {2015 Jan 5}, pages = {80-6}, abstract = {

At the onset of each flight bout in flies, neural circuits in the CNS must rapidly integrate multimodal sensory stimuli and synchronously engage hinges of the left and right wings for coordinated wing movements. Whereas anatomical and physiological investigations of flight have been conducted on larger flies, molecular genetic studies in Drosophila have helped identify neurons that mediate various levels of flight control. However, neurons that might mediate bilateral coordination of wing movements to precisely synchronize left and right wing engagement at flight onset and maintain their movement in perfect coordination at rapid frequencies during flight maneuvers remain largely unexplored. Wing coordination could be directly modulated via bilateral sensory inputs to motoneurons of steering muscles and/or through central interneurons. Using a Ca(2+)-activity-based GFP reporter, we identified three flight-activated central dopaminergic interneurons in the ventral ganglion, which connect to and activate motoneurons that innervate a pair of direct-steering flight muscles. The activation of these newly identified dopaminergic interneurons is context specific. Whereas bilateral wing engagement for flight requires these neurons, they do not control unilateral wing extension during courtship. Thus, independent central circuits function in the context of different natural behaviors to control the motor circuit for Drosophila wing movement.

}, keywords = {Animals, Central Nervous System, Dopaminergic Neurons, Drosophila, Flight, Animal, Genes, Reporter, Male, Synapses, Wings, Animal}, issn = {1879-0445}, doi = {10.1016/j.cub.2014.10.069}, author = {Sadaf, Sufia and Reddy, O Venkateswara and Sane, Sanjay P and Hasan, Gaiti} } @article {1033, title = {Store-Operated Calcium Entry through Orai Is Required for Transcriptional Maturation of the Flight Circuit in Drosophila.}, journal = {J Neurosci}, volume = {35}, year = {2015}, month = {2015 Oct 7}, pages = {13784-99}, abstract = {

UNLABELLED: Store operated calcium entry (SOCE) is thought to primarily regulate calcium homeostasis in neurons. Subsequent to identification of Orai as the SOCE channel in nonexcitable cells, investigation of Orai function in neurons demonstrated a requirement for SOCE in Drosophila flight. Here, by analysis of an Orai mutant and by controlled expression of a dominant-negative Drosophila Orai transgene, we show that Orai-mediated SOCE is required in dopaminergic interneurons of the flight circuit during pupal development. Expression of dominant-negative Orai in dopaminergic neurons of pupae abolished flight. The loss of Orai-mediated SOCE alters transcriptional regulation of dopaminergic neurons, leading to downregulation of the enzyme tyrosine hydroxylase, which is essential for dopamine synthesis, and the dopamine transporter, which is required for dopamine uptake after synaptic release. These studies suggest that modulation of SOCE could serve as a novel mechanism for restoring dopamine levels in dopaminergic neurons.

SIGNIFICANCE STATEMENT: The specificity of an animal{\textquoteright}s response to an environmental stimulus is determined in part by the release of neurotransmitters, which are sensed by responding neurons through cognate receptors on their surface. One way by which neurons respond is through release of calcium from intracellular stores followed by store refilling from extracellular calcium sources. This mechanism is called store-operated calcium entry (SOCE). The function of SOCE in neurons has been debated. Here we describe a new function for SOCE in the regulation of neurotransmitter levels in Drosophila flight neurons. This cell-signaling mechanism is required to maintain optimal levels of a key enzyme for dopamine synthesis and may serve as a mechanism for restoring dopamine levels in relevant pathological conditions.

}, issn = {1529-2401}, doi = {10.1523/JNEUROSCI.1680-15.2015}, author = {Pathak, Trayambak and Agrawal, Tarjani and Richhariya, Shlesha and Sadaf, Sufia and Hasan, Gaiti} }