Transmembrane Protein Ttyh1 Regulates The Characteristics Of Neural Stem Cells Through Calcium Signaling Pathway

【Background】Neural stem cells（NSCs）have the ability of self-renewal,and can give rise to multipotent progenitors and finally differentiate into neurons,astrocytes and oligodendrocytes by symmetric division or asymmetric division.Neuroepithelial cells which participate in the development of the central nervous system are considered primitive neural stem cells in the embryonic brain.Neuroepithelial cells can amplify themselves by symmetric division.As the development of the fetal cerebral cortex,neuroepithelial cells gradually transform into radial glial cells which also have the characteristics of neural stem cells.During this period,radial glial cells can produce a newborn radial glial cell and a neuron cell by asymmetric division,and the newly born neuron can migrate to the surface of cerebral cortex along radial glial fibers.In the adult brain,neural stem cells are GFAP+SOX2+cells exist in the subventricular zone（SVZ）subjacent to the ependymal layer and the subgranular zone（SGZ）of the hippocampal dentate gyrus,and neurogenesis occurs throughout adult life in these regions.Forebrain ependymal cells which derived from radial glial cells in the embryonic brain have been considered to be neural stem cells.Forebrain ependymal cells also express some genes rich in neural stem cells,such as CD133、Nestin and so on.It has been indicated that ependymal cells keep quiescent under normal condition.However,ependymal cells gave rise to DCX+neuroblasts involved in the repairing progress in response to stroke.The tweety genes in vertebrates contains three family members:Ttyh1,Ttyh2 and Ttyh3,which are highly conserved during evolution,and recent studies have shown tweety genes are involved in a variety of cellular and molecular behaviors such as cell division,cell adhesion,calcium regulation.It has been confirmed that Tweety family genes are associated with the development of various diseases.In the animal model of epilepsy,Ttyh1 is up-regulated in the inner layer of the hippocampal dentate gyrus.Ttyh1was also confirmed to be a potent regulator of tumor-cell colonization and proliferation in gliomas.While Ttyh2 is highly expressed in colon cancer and renal carcinoma,Ttyh3 is involved in the calcium dynamics of excitable tissues such as skeletal muscle,brain and spinal cord.By in situ hybridization assay and immunofluorescence staining,it was found that Ttyh1 was specifically expressed in the central nervous system,especially in the ventricular and subventricular zone where neural stem cells were distributed.Therefore,Ttyh1 is likely to be involved in the regulation of proliferation and differentiation of neural stem cells.Ca²⁺is a dynamic cellular secondary messenger which plays a significant role in the development of the vertebrate nervous system.The recent discovery of Endoplasmic Reticulum-driven store-operated Ca²⁺entry（SOCE）indicates Ca²⁺sigaling mediates a array of cellular progress in neural stem cell,including cell proliferation,neural differentiation.SOCE is based on the interaction of ER Ca²⁺sensor STIM1 and the highly Ca²⁺selective channel Orai1.When ER Ca²⁺content is high,Ca²⁺are bound to the EF hands of STIM1 and make the proteins inactive.Decreasing in ER Ca²⁺concentration leads to the dissociation of Ca²⁺from the STIM EF hands,resulting in the aggregation of STIM1 and activate the highly Ca²⁺-selective channel Orai1.Recent studies demonstrated that the knockdown of SOCE in mouse NPCs reduced cell proliferation in the SVZ of adult mouse brains.Moreover,knockdown of STIM1 in human neural precursor cell results in decreased cell proliferation and increased neural differentiation.The aspartic acid-rich C-terminal portion of Ttyh1 has been proved to possess Ca²⁺-binding activity.Our previous results has confirmed that Ttyh1 is specifically localized on the endoplasmic reticulum,suggesting Ttyh1 exists as a kind of calcium-binding protein on the endoplasmic reticulum which is involved in regulating the cellular calcium level.The423-433 amino acids of Ttyh1 which bound to Ca²⁺are confirmed to be the key protein position affecting neural stem cell properties.Therefore,we hypothesized that Ttyh1controls neural stem cell characteristics by regulating cellular calcium levels.【Objective】We determined to specific the distribution of Ttyh1 in the telencephalon by immunofluorescence staining using Ttyh1 antibodies,and to explore its main functions in the process of proliferation and differentiation of neural stem cells by Ttyh1 siRNA technology.Besides,we aim to find the changes in neural stem cell niche after Ttyh1knockout in mouse.Finally,we aim to investigate the mechanism of Ttyh1 in regulating the characteristics of neural stem cells.【Methods】1.In order to specific the distribution of Ttyh1,immunofluorescence staining was performed using Ttyh1 antibody which was constructed by our research team in previous time.2.The si-RNA of Ttyh1 was constructed,and the phenotypic changes of neural stem cells were observed after Ttyh1 knockdown in vitro.3.Ttyh1 knockout mice were constructed by CRISPR-Cas9 technology,and changes in proliferation and differentiation of neural stem cells were observed.4.We performed RNA-seq analysis on neural stem cells derived from E14.5 Ttyh1KO mice and littermate controls in order to explore the mechanism of Ttyh1 in neural stem cells.【Results】1.Immunofluorescence staining results showed that Ttyh1 is highly expressed in ventricular and subventricular zone（V-SVZ）where neural stem cells are located in both embryonic and postnatal telencephalon.The majority of Ttyh1+cells co-expressed with CD133,a quiescent stem cell marker.Whereas none of Ttyh1+cells co-expressed with Ki67,suggesting that Ttyh1+cells were not actively dividing.2.Transient increased sphere formation ability was observed after Ttyh1 knockdown in NSCs,Whereas ultimately leads to the decrease in stemness of neural stem cells,suggesting that Ttyh1 played important roles in self-renewal and proliferative activities of NSCs.In differentiation experiment of neural stem cells,more Ttyh1-deficient NSCs underwent multilineage differentiation into MAP2+neuronal precursor cells,O4+oligodendrocytes in contrast to control NSCs.3.By long-term BrdU labeling in Ttyh1 knockout mice,we found that Ttyh1 knockout resulted in a decrease in the migration capacity of BrdU+neural stem cells.Therefore,more BrdU+neural stem cells retained in the ventricular and subventricular zone.Moreover,the immunofluorescence staining results showed that loss of Ttyh1 lead to a transient increase in neurogenesis in Ttyh1 KO mouse.But the number of GFAP+SOX2+quiescent neural stem cells and S100β+ependymal cells were depleted early in the stem cell niche.4.We performed RNA-seq analysis on neural stem cells derived from E14.5 Ttyh1KO mice and littermate controls.The RNA-seq analysis results showed that Ttyh1 knockout leads to changes in molecules associated with Endoplasmic Reticulum-driven store-operated Ca²⁺entry（SOCE）,such as STIM1 and Orai1,suggesting that Ttyh1 may influence the characteristics of neural stem cells by regulating the calcium signaling pathway.5.Ttyh1 expression was decreased after blocking Notch signaling pathway by GSI and increased after Notch activation in turn.We further confirmed the positive regulation of Notch signaling on Ttyh1 through reporter gene experiments.【Conclusions】1.Ttyh1 is highly expressed in ventricular and subventricular zone（V-SVZ）where neural stem cells are located in both embryonic and postnatal telencephalon.In postnatal telencephalon,Ttyh1 is mainly expressed in CD133+quiescent neural stem cells.Whereas none of Ttyh1+cells were co-expressed with Ki67+,a marker of dividing cells.2.Ttyh1 is involved in the regulation of proliferation and differentiation of neural stem cells.After Ttyh1 knockout,quiescent neural stem cells are not properly maintained and mostly become proliferative cells,leading to a transient increase in stemness of neural stem cells.Loss of Ttyh1 also result in transient increase in neurogenesis in Ttyh1 KO mouse.But the long-term result of the depletion of quiescent neural stem cells eventually leads to decline of neurogenesis.3.The results suggested that Ttyh1 exist as a kind of calcium-binding protein on the endoplasmic reticulum,and regulate neural stem cell characteristics through calcium signaling pathway.4.The expression of Ttyh1 in neural stem cells is regulated by Notch signaling pathway.After blocking Notch signaling pathway,the expression of Ttyh1 in neural stem cells is significantly reduced.Notch signaling pathway can participate in the regulation of neural stem cells by regulating the expression of Ttyh1.