@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 {93671, title = {Pluripotent Stem Cell Derived Neurons as In Vitro Models for Studying Autosomal Recessive Parkinson{\textquoteright}s Disease (ARPD): PLA2G6 and Other Gene Loci.}, journal = {Adv Exp Med Biol}, year = {2021}, month = {2021 May 15}, abstract = {

Parkinson{\textquoteright}s disease (PD) is a neurodegenerative motor disorder which is largely sporadic; however, some familial forms have been identified. Genetic PD can be inherited by autosomal, dominant or recessive mutations. While the dominant mutations mirror the prototype of PD with adult-onset and L-dopa-responsive cases, autosomal recessive PD (ARPD) exhibit atypical phenotypes with additional clinical manifestations. Young-onset PD is also very common with mutations in recessive gene loci. The main genes associated with ARPD are Parkin, PINK1, DJ-1, ATP13A2, FBXO7 and PLA2G6. Calcium dyshomeostasis is a mainstay in all types of PD, be it genetic or sporadic. Intriguingly, calcium imbalances manifesting as altered Store-Operated Calcium Entry (SOCE) is suggested in PLA2G6-linked PARK 14 PD. The common pathways underlying ARPD pathology, including mitochondrial abnormalities and autophagic dysfunction, can be investigated ex vivo using induced pluripotent stem cell (iPSC) technology and are discussed here. PD pathophysiology is not faithfully replicated by animal models, and, therefore, nigral dopaminergic neurons generated from iPSC serve as improved human cellular models. With no cure to date and treatments aiming at symptomatic relief, these in vitro models derived through midbrain floor-plate induction provide a platform to understand the molecular and biochemical pathways underlying PD etiology in a patient-specific manner.

}, issn = {0065-2598}, doi = {10.1007/5584_2021_643}, author = {Gopurappilly, Renjitha} } @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 {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 {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 {22813, title = {Transcriptional profiling of human neural precursors post alcohol exposure reveals impaired neurogenesis via dysregulation of ERK signaling and miR-145.}, journal = {J Neurochem}, volume = {146}, year = {2018}, month = {2018}, pages = {47-62}, abstract = {

Gestational alcohol exposure causes a range of neuropsychological disorders by modulating neurodevelopmental genes and proteins. The extent of damage depends on the stage of the embryo, as well as dosage, duration, and frequency of exposure. Here, we investigated the neurotoxic effects of alcohol using Human Embryonic Stem Cells (hESC). Multiple read-outs were engaged to assess the proliferation and differentiation capacity of neural precursor cells (NPC) upon exposure to 100 mM ethanol for 48 hrs corresponding to the blood alcohol levels for binge drinkers. Whole genome analysis revealed a spatiotemporal dysregulation of neuronal and glial specific gene expression that play critical roles in Central Nervous System (CNS) development. Alterations observed in the transcriptome may be attributed to epigenetic constitution witnessed by differential histone H3 Lys-4/Lys-27 modifications and acetylation status. In-depth mRNA and protein expression studies revealed abrogated ERK signaling in alcohol- treated cells. Consistent with this finding, Ingenuity Pathway Analysis (IPA) and microRNA profiling demonstrated upregulation of miR-145 by targeting the neural specifier Sox-2. We also show that the neurite branching complexity of TUJ1+ neurons was greatly reduced in response to alcohol. Lastly, in vivo studies using zebrafish embryos reconfirmed the in vitro findings. Employing molecular endpoints in a human model, this report indicates for the first time that acute alcohol exposure could lead to impaired brain development via perturbation of ERK pathway and miR-145. However, it still needs to be addressed whether these modulations sustain throughout development, compromising the ability of the individual during adulthood and aging. This article is protected by copyright. All rights reserved.

}, issn = {1471-4159}, doi = {10.1111/jnc.14155}, author = {Louis, Lithin K and Gopurappilly, Renjitha and Surendran, Harshini and Dutta, Sunit and Pal, Rajarshi} }