IP3Rs and nSOCE-Tied Together at Two Ends.

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 Ca2+ signaling is one such mechanism where extracellular signals received at the cell membrane communicate with endoplasmic reticular stores of Ca2+, to stimulate appropriate Ca2+-mediated changes in cellular physiology. The amplitude and dynamics of endoplasmic reticulum (ER)-Ca2+ release in response to extracellular signals determines the nature of the cellular response. An understanding of how ER-Ca2+ channels might regulate cellular Ca2+ 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.

Orai-mediated calcium entry determines activity of central dopaminergic neurons by regulation of gene expression.

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 Ca2+ signals, through the store-operated Ca2+ 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 Ca2+ release through the endoplasmic reticulum (ER) localised inositol 1,4,5-trisphosphate receptor followed by ER-store depletion and Orai-mediated store-operated Ca2+ entry (SOCE). Analysis of gene expression in fpDANs followed by genetic, cellular, and molecular studies identified Orai-mediated Ca2+ 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 Set2, 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.

ER-Ca2+ stores and the regulation of store-operated Ca2+ entry in neurons.

Key components of endoplasmic reticulum (ER) Ca2+ release and store-operated Ca2+ entry (SOCE) are likely expressed in all metazoan cells. Due to the complexity of canonical Ca2+ entry mechanisms in neurons, the functional significance of ER-Ca2+ release and SOCE has been difficult to identify and establish. In this review we present evidence of how these two related mechanisms of Ca2+ signalling 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 Ca2+ homeostasis can have strongly detrimental effects on neurons, leading to cell death, it is essential that neuronal SOCE is carefully regulated. We go on to discuss some mechanisms of SOCE regulation that have been identified in Drosophila and mammalian neurons. These include specific splice variants of stromal interaction molecules, different classes of membrane-interacting proteins and an ER-Ca2+ 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.

Regulation of store-operated Ca2+ entry by IP3 receptors independent of their ability to release Ca2.

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.

A STIM dependent dopamine-neuropeptide axis maintains the larval drive to feed and grow in Drosophila.

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') 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' 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' neurons. Optogenetic stimulation of THD' neurons activated neuropeptidergic cells, including median neuro secretory cells that secrete insulin-like peptides. Loss of STIM in THD' 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.

Generation of feeder-independent transgene-free iPSC lines from a young-onset Parkinson's disease (YOPD) patient with a homozygous PLA2G6: c.2222G>A (p. Arg741Gln) mutation (NCBSi003-A) and unaffected heterozygous parent (NCBSi004-A).

Phospholipase A2 group 6 (PLA2G6, iPLA2ß or PARK14) gene encodes a calcium-independent group 6 phospholipase A2 enzyme and is associated with young-onset autosomal recessive Parkinson'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.

Store-operated Ca2+ entry regulates neuronal gene expression and function.

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.

Two photon imaging of calcium responses in murine Purkinje neurons.

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 Ca2+ transients can be challenging. Here, we describe a protocol for measuring Ca2+ transients in PNs from adult mice, including problem-solving tips. This protocol can be used to measure neuronal excitability and agonist-mediated Ca2+ signaling in cerebellar slices expressing a genetic Ca2+ 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).

IP3-mediated Ca2+ signals regulate larval to pupal transition under nutrient stress through the H3K36 methyltransferase Set2.

Persistent loss of dietary protein usually signals a shutdown of key metabolic pathways. In Drosophila larvae that have reached a 'critical weight' 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.

Purkinje Neurons with Loss of STIM1 Exhibit Age-Dependent Changes in Gene Expression and Synaptic Components.

The stromal interaction molecule 1 (STIM1) is an ER-Ca2+ sensor and an essential component of ER-Ca2+ store operated Ca2+ entry. 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 led to neurodegenerative conditions, as evident in individuals with mutations of the ER-Ca2+ channel, the inositol 1,4,5-triphosphate receptor. Here, we asked whether changes in such intrinsic neuronal properties, because of loss of STIM1, have an age-dependent impact on PNs. Consequently, we analyzed mRNA expression profiles and cerebellar morphology in PN-specific STIM1 KO 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 among others. Gene expression changes correlated with altered patterns of dendritic morphology and greater innervation of PN dendrites by climbing fibers, in aging 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 STATEMENT In 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 is 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.

Deficits Associated With Loss of STIM1 in Purkinje Neurons Including Motor Coordination Can Be Rescued by Loss of Septin 7.

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 Ca2+ entry (SOCE) channel, Orai in Drosophila neurons, and in human neural progenitor cells. Knockdown of STIM, a Ca2+ sensor in the endoplasmic reticulum (ER) and an integral component of SOCE, leads to flight deficits in Drosophila 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 SEPT7 gene in STIM1 KO 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 STIM1 KO animals. Our findings are relevant in the context of identifying SEPT7 as a putative therapeutic target for various neurodegenerative diseases caused by reduced intracellular Ca2+ signaling.

Modulation of flight and feeding behaviours requires presynaptic IP3Rs in dopaminergic neurons.

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. Drosophila flight is one such innate behaviour that is modulated by intracellular calcium release through inositol 1,4,5-trisphosphate receptors (IP3Rs). Cellular mechanism(s) by which IP3Rs modulate neuronal function for specific behaviours remain speculative, in vertebrates and invertebrates. To address this, we generated an inducible dominant negative form of the IP3R (IP3RDN). Flies with neuronal expression of IP3RDN exhibit flight deficits. Expression of IP3RDN helped identify key flight-modulating dopaminergic neurons with axonal projections in the mushroom body. Flies with attenuated IP3Rs 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.

SEPT7 regulates Ca2+ entry through Orai channels in human neural progenitor cells and neurons.

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 Ca2+ entry through the store-operated Ca2+ 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 Ca2+ entry via Orai, in the absence of ER-Ca2+ 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-Ca2+ sensor STIM1 observed near the plasma membrane in SEPT7 KD hNPCs, supports the idea that Septin7 containing heteromers prevent Ca2+ 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.

Surviving nutritional deprivation during development: neuronal intracellular calcium signaling is critical.

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 Ca2+ signaling arising from the release of Ca2+ stored in the endoplasmic reticulum (ER), which triggers store-operated Ca2+ entry. We describe a role for ER-store Ca2+ 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 2+ 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 Ca2+ for neuronal health, with consequences for animal development.

Ral function in muscle is required for flight maintenance in Drosophila.

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.

Implications of the Sap47 null mutation for synapsin phosphorylation, longevity, climbing proficiency and behavioural plasticity in adult Drosophila.

The Sap47 gene of Drosophila melanogaster encodes a highly abundant 47 kDa synaptic vesicle-associated protein. Sap47 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 Drosophila synapsin has repeatedly been shown to be important for behavioural plasticity but it was not known where these phospho-synapsin isoforms are localized in the brain. 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. The effects of knockout of Sap47 or synapsin on olfactory associative learning/memory support the hypothesis that both proteins operate in the same molecular pathway. We therefore asked if this might also be true for other aspects of their function. We show that knockout of Sap47 but not synapsin reduces lifespan, whereas knockout of Sap47 and synapsin, either individually or together, affects climbing proficiency, 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 Sap47 and synapsin 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.

Measurement of Store-Operated Calcium Entry in Human Neural Cells: From Precursors to Differentiated Neurons.

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.

ER-Ca2+ sensor STIM regulates neuropeptides required for development under nutrient restriction in Drosophila.

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.

Extended Flight Bouts Require Disinhibition from GABAergic Mushroom Body Neurons.

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.

IP3 receptors and Ca2+ entry.

Inositol 1,4,5-trisphosphate receptors (IP3R) are the most widely expressed intracellular Ca2+ release channels. Their activation by IP3 and Ca2+ allows Ca2+ to pass rapidly from the ER lumen to the cytosol. The resulting increase in cytosolic [Ca2+] may directly regulate cytosolic effectors or fuel Ca2+ uptake by other organelles, while the decrease in ER luminal [Ca2+] stimulates store-operated Ca2+ entry (SOCE). We are close to understanding the structural basis of both IP3R activation, and the interactions between the ER Ca2+-sensor, STIM, and the plasma membrane Ca2+ channel, Orai, that lead to SOCE. IP3Rs are the usual means through which extracellular stimuli, through ER Ca2+ release, stimulate SOCE. Here, we review evidence that the IP3Rs most likely to respond to IP3 are optimally placed to allow regulation of SOCE. We also consider evidence that IP3Rs may regulate SOCE downstream of their ability to deplete ER Ca2+ stores. Finally, we review evidence that IP3Rs in the plasma membrane can also directly mediate Ca2+ entry in some cells.