Stable STIM1 Knockdown in Self-Renewing Human Neural Precursors Promotes Premature Neural Differentiation

Abstract

Ca2+ 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 Ca2+ 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 Ca2+ 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 STIM1 knockdown. Interestingly, pathways down-regulated through STIM1 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 STIM1 knockdown. The STIM1 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 STIM1 mediated SOCE in human NPCs regulates gene expression changes, that in vivo are likely to physiologically modulate the self-renewal and differentiation of NPCs.

In vitro human stem cell derived cultures to monitor calcium signaling in neuronal development and function

“…SOCE is one of these processes and is triggered by G-protein coupled PLC activation, a signalling mechanism used by several cell surface receptors that are expressed in the human cerebral cortex and reported to be important for brain function [e.g metabotropic glutamate receptor] and PLC signalling has also been linked to several brain disorders 29 . Studies in murine neural precursor cells (NPCs) have shown robust SOCE and depletion of STIM1 or ORAI1, key elements of SOCE results in defective NPC proliferation 30 ; a recent study has also suggested a conserved role for SOCE in human NPC proliferation 31 . In this study we developed protocols to monitor SOCE during in vitro differentiation and found a dynamic pattern of SOCE activity.…”

STIM Proteins and Glutamate Receptors in Neurons: Role in Neuronal Physiology and Neurodegenerative Diseases

“…Both the suppression and deletion of STIM1 or Orai1 significantly alleviate the proliferation of embryonic and adult NPCs [53]. STIM1 knockdown also decreases the proliferation and early differentiation of human NPCs [54]. In addition to Orai activation, STIM proteins may induce Ca 2+ influx via TRPCs [5,55].…”

Dysregulation of Neuronal Calcium Signaling via Store-Operated Channels in Huntington's Disease

“…Furthermore, STIM1 protein is responsible for mGluR1-dependent synaptic transmission in cerebellar Purkinje neurons (Hartmann et al, 2014 ). Accumulating evidence indicates a role for STIM1 in neurogenesis (Somasundaram et al, 2014 ) and the proliferation and early differentiation of neural progenitor cells (NPCs) (Somasundaram et al, 2014 ; Gopurappilly et al, 2018 ). The knockdown of both STIM isoforms reduced SOCE and inhibited the entry of mouse embryonic stem cells into a neural lineage (Hao et al, 2014 ).…”