@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 {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 {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} }