Nuclear Orphan Receptor TLX Induces Oct-3/4 for the Survival and Maintenance of Adult Hippocampal Progenitors upon Hypoxia

Abstract

Hypoxia promotes neural stem cell proliferation, the mechanism of which is poorly understood. Here, we have identified the nuclear orphan receptor TLX as a mediator for proliferation and pluripotency of neural progenitors upon hypoxia. We found an enhanced early protein expression of TLX under hypoxia potentiating sustained proliferation of neural progenitors. Moreover, TLX induction upon hypoxia in differentiating conditions leads to proliferation and a stem cell-like phenotype, along with coexpression of neural stem cell markers. Following hypoxia, TLX is recruited to the Oct-3/4 proximal promoter, augmenting the gene transcription and promoting progenitor proliferation and pluripotency. Knockdown of Oct-3/4 significantly reduced TLX-mediated proliferation, highlighting their interdependence in regulating the progenitor pool. Additionally, TLX synergizes with basic FGF to sustain cell viability upon hypoxia, since the knockdown of TLX along with the withdrawal of growth factor results in cell death. This can be attributed to the activation of Akt signaling pathway by TLX, the depletion of which results in reduced proliferation of progenitor cells. Cumulatively, the data presented here demonstrate a new role for TLX in neural stem cell proliferation and pluripotency upon hypoxia.The adult brain retains a reservoir of stem progenitor cells in the hippocampal "neurogenic zone" capable of proliferative activity throughout life (1). These undifferentiated precursors that retain the ability to proliferate and self-renew can give rise to both neuronal and glial lineages (2). Recent studies have emphasized the role of hypoxia in maintaining pluripotency and increased proliferation of neural stem cells (3-5).However, the molecular mechanisms underlying the increased proliferation and pluripotency of neural stem cells are yet unexplored. TLX (NR2E1), an orphan nuclear receptor expressed in vertebrate forebrains (6), is an essential regulator of adult neural stem cell self-renewal (7). TLX maintains neural stem cells in an undifferentiated and self-renewable state by complexing with histone deacetylases to repress TLX downstream target genes, such as p21 and Pten, promoting cellular proliferation (8). Furthermore, TLX is expressed in the subventricular neural stem cells in embryonic brains and plays an important role in neural development by regulating cell cycle progression of neural stem cells (9 -11). Also, TLXpositive neural stem cells play an important role in spatial learning and memory in adult brains (12).This study aimed to investigate the role of TLX in promoting neural progenitor population under differentiating conditions, considering the fact that TLX acts as a hypoxic sensor in retinal astrocytes (13), and hypoxia promotes proliferation/ dedifferentiation of progenitor cells. Our results demonstrate that TLX is responsive to hypoxia in both differentiating and proliferating conditions. The knockdown of TLX attenuates hypoxia-mediated progenitor proliferation and induces differentiation. Further investigatio...

The Orphan Nuclear Receptor TLX/NR2E1 in Neural Stem Cells and Diseases

“…In the mouse, multiple genes involved in the development of the CNS and visual system have been identified as Tlx targets, such as Gsh2, Pax2, Pten, p21, p57, S100b, Aqp4, Plce1, Wnt7a, microRNA-9, Bmp4, and GFAP [21,[32][33][34][35][36][37][45][46][47][48][49][50] (Table 1). Interestingly, Tlx may act as a transcriptional activator for the expression of sirt1, Wnt7a, Mash1, and Oct-3/4 based on in vitro luciferase assays and chromatin immunoprecipitation assays [16,21,[51][52][53]. A consensus Nr2e1 binding sequence within the promoter region of its target gene has been proposed to contain AAGTCA, which has been identified in the promoters of Wnt7a, GFAP, S100b, Oct3/ 4, p21, Pax2, Plce1, and Pten [21,32,34,45,46,53,54].…”

“…Interestingly, Tlx may act as a transcriptional activator for the expression of sirt1, Wnt7a, Mash1, and Oct-3/4 based on in vitro luciferase assays and chromatin immunoprecipitation assays [16,21,[51][52][53]. A consensus Nr2e1 binding sequence within the promoter region of its target gene has been proposed to contain AAGTCA, which has been identified in the promoters of Wnt7a, GFAP, S100b, Oct3/ 4, p21, Pax2, Plce1, and Pten [21,32,34,45,46,53,54]. Given the functional equivalence and the highly-conserved DBDs between human TLX and mouse Tlx [13,14], TLX may have the capacity to bind a similar consensus promoter sequence and suppress or activate the expression of multiple human counterparts of the identified mouse Tlx targets.…”

TLX: A master regulator for neural stem cell maintenance and neurogenesis

“…TLX binds a highly conserved 5′-AAGTCA-3′ motif to promote or repress target gene expression [16]. The TLX targets Ascl1 , Pou5f1 , Pax2 , Mir9 , Mir137 , Pten , Cdkn1a , Sirt1 , and Wnt7a were each identified through DNA binding assays or conserved motif analyses [11, 12, 16, 26, 27, 33, 35, 45–49]. …”

“…Interestingly, TLX balances dichotomous roles under hypoxic conditions, the regulation of NSC commitment to the neuronal lineage by Ascl1 and the maintenance of proliferating neural progenitor pools through the fine-tuning of Pou5f1 ( Oct3/4 ) expression [26, 27, 45]. In this second role, TLX serves as a critical mediator of hypoxia-induced NSC proliferation and neural progenitor pluripotency.…”

Hypoxia in the regulation of neural stem cells

“…In fact, in different stem cell systems, mild hypoxia activates molecular pathways that regulate Wnt/ beta-catenin, Oct4 and Notch signaling, all positive regulators of self-renewal and proliferation [28][29][30][31][32]. Under mild hypoxia, embryonic stem cells (ESCs) [33] as well as somatic adult stem cells, e.g., mesenchymal stem cells [34,35], hematopoietic stem cells [36] and NSCs [37,38], all tend to proliferate and self-maintain.…”