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'''Role of PARPs and NAD+ Metabolism in Early Embryogenesis and Pluripotency '''

Introduction
Several groups studying pluripotency have utilized XAV939, an inhibitor of Poly ADP-ribose Polymerase (PARP) proteins, to stabilize alternative human and murine pluripotent states in combination with GSK-3β co-inhibition [1-4]. The combined utilization of XAV939 and GSK-3β inhibition—via the use of the chemical molecule CHIR99021—in primed murine Epiblast stem cells and primed human Embryonic Stem Cells (hESCs) increased WNT signaling and AXIN expression, while simultaneously stabilizing the AXIN-catenin complex. The result of this stabilization increased cytoplasmic localization of the active non-phosphorylated isoform of β-catenin [1]. In 2016, Zimmerlin et al [2] reported that the combined utilization of XAV939 and CHIR99021 with simultaneous MEK inhibition reverted conventional hESCs to an epigenetically and functionally novel state closely mimicking the human pre-implantation pluripotent state referred to as naïve pluripotency. Subsequent groups have also now shown that XAV939 is crucial for the stabilization of earlier and more functionally robust pre-implantation pluripotent states (i.e expanded potential stem cells, totipotent-like stem cells) [4-5]. Altogether these insights point to the importance of tankyrases, and PARPs in general, in the maintenance of early embryonic functionality.

Background
There are 17 proteins involved in various cellular processes, including chromatin remodeling, stress response, DNA repair, and apoptosis that together form the PARP family. The most characterized and well-known member of the PARP family is that of PARP1[6]. PARP1 was first identified for its role in the detection and repair of single-strand DNA breaks. Subsequent studies have shown that PARP1 may play a role in alternative DNA repair pathways, including non-homologous end joining, nucleotide excision repair, homologous recombination, and DNA mismatch repair. Because of PARP’s role in the regulation of DNA repair pathways, most notably by the regulation of BRCA1 and BRCA2, PARP inhibitors (PARPi) have been utilized as novel anti-cancer therapeutics. These include the treatments of ovarian, breast and pancreatic cancers with BRCA mutations [1,6]. PARPs are enzymatic proteins that cause PolyADP-ribosylation, also known as PARylation. Parylation is a post-translational modification event whereby polymers of ADP-ribose (poly(adenosinediphosphate-ribose)) are covalently attached to proteins by PARP [1]. Crucially, PARPs utilize the metabolite NAD+ in their enzymatic cleavage cite as a substrate for PARylation [7]. By PARylating itself, as well as histones and other DNA-binding proteins, PARPs can regulate protein localization, abundance, and enzymatic activity [1,7]. An important role of PARylation in embryonic development was first shown in a study in 2001, where it was observed that uncontrolled accumulation of poly-ADP-ribose resulted in early embryonic lethality [7-9]. The role of PARylation in regulating WNT signaling was subsequently seen in pluripotent cells and cancer cell survival studies using PARPi. XAV939, one of the PARPi used in these studies, was first identified as an inhibitor of WNT signaling through its ability to induce β-catenin degradation via antagonism of the PARylation activity of tankyrases (members of the PARP family) [1]. Further characterization of the small molecule XAV939 showed that it inhibited WNT signaling in cancer cells by stabilizing AXIN and bolstering the β-catenin destruction complex [7]. XAV939 binds to a conserved subsite within the nicotinamide pocket of the PARP enzymatic domain binding groove of PARP family proteins [1,7].



Metabolism of the Early Embryo
The newly fertilized mammalian embryo transitions between different waves of oxygen-dependent metabolism pathways largely correlating with the waves of functional potency of their cell types [1]. After zygotic genome activation blastomeres represent a totipotent functional capacity able to produce both the embryonic and extraembryonic tissues of the fetus [8]. As the embryo transitions out of the fallopian tube and into the uterus, blastomeres become more lineage restricted and biased toward producing extraembryonic or embryonic lineages [8]. This skewing is complete at the morula compaction stage in the murine embryo. Finally, the inner cell mass (ICM) of the pre-implantation blastocyst contains a more potent cell population that differs from its post-implantation counterpart [8]. As referred to earlier, the pre-implantation ICM exist in a naïve or ground pluripotent state, whereas post-implantation ICM exist in a primed state [8]. Stable embryonic stem cells (ESCs) derived from either pluripotent state has given insights into the metabolism of the early mammalian embryo. Pre-implantation blastocyst derived naïve ESCs largely utilize glycolysis and oxidative phosphorylation (OXPHOS) [1]. After implantation, the blastocyst ICM then transitions to glycolysis dependence in the primed state even though primed ESCs contain functionally mature mitochondria [1,10]. This transition is coordinated by the transcription factors Zic3 and Esrrb in naïve ESCs [10]. Naïve ESCs also have a more robust glycolytic output compared with primed ESCs [10]. Many have suggested that this divergent metabolic pathway in naïve versus primed pluripotent stem cells is not only crucial for understanding differences in ATP production, but also could play a role in shifts in early embryo transcriptional machinery and chromatin accessibility [1,10].

Role NAD+ Metabolism in Regulating Early Pluripotency
NAD+ has been shown to increase hESC mitochondrial oxidative metabolism and stimulate amino acid turnover—increasing glutamine consumption— while also attenuating glycolysis [11]. The regulation of NAD+ recycling via malate aspartate shuttle activity is crucial for hESC self-renewal, as it is responsible for 80% of the oxidative capacity of hESC mitochondria [11]. Additionally, cellular NAD+ levels can be directly influenced via the metabolic state of cells which can impact the activity of NAD+-dependent histone deacetylase sirtuins, including SIRT1 [1]. SIRT1 is highly expressed in hESCs and has been shown to control cell fate by regulating the crucial pluripotency factor NANOG [10]. During hESC differentiation, SIRT1 is downregulated, which causes the acetylation and reactivation of multiple somatic-lineage differentiation genes [10]. Furthermore, NAD+ has been shown to increase other hESC pluripotency marker expression as well as reduce global histone 3 lysine 27 trimethylation (H3K27me3) levels on chromatin [11].

The switch between bivalent respiration (i.e. glycolysis and OXPHOS) to exclusive anaerobic glycolysis in the murine and human blastocyst also further results in differential regulation of other epigenetic regulators other than SIRT [1]. This includes nicotinamide-N-methyltransferase (NNMT), an enzyme which consumes two substrates, S-adenosyl methionine (SAM) and nicotinamide [1]. Sperber et al [12] proposed a mechanism in which naïve pluripotent cells are regulated by an NNMT-induced methyl-sink. In the NNMT-induced methyl-sink, NNMT and its enzymatic product 1–1-methyl-nicotinamide (1-MNA) are upregulated, while levels of its substrates, nicotinamide and SAM are reduced. This would thereby cause a sequestering of histone repressive marks such as H3K27me3 and H3k9me3 [12]. Thus NAD+ metabolite level may play an important role in regulating the epigenetic state of pluripotent cells.

Role of PARPs in Regulating NAD+ and Metabolism


As stated earlier, TNKS1 and TNKS2 utilize NAD+ as a substrate to post-translationally modify proteins, resulting in divergent protein binding affinity or recruitment of the E3 ubiquitin-protein ligase RNF146 for proteolysis [1]. Additionally, PARylation causes a significant reduction in cellular NAD+ and ATP levels [13]. This depleted ATP pool due to PARP activity can be disastrous to the cell, leading to mitochondrial energy failure and embryonic lethality [1, 7, 9]. Despite high sequence conservation between the enzymatic domains of PARPs, PARP-1, PARP-2, and tankyrases have been shown to produce longer PAR polymers. Thus, these proteins (PARP1, PARP2, tankyrases) are responsible for most of PARP-related NAD+ consumption [13]. Because of the use of the XAV939 small molecule in cell culture conditions to stabilize early pre-implantation functional states, it has been postulated that the NAD+ abundance is crucial for the early embryo [1]. Even so, more work is required in our understanding of Tankyrases role in ESC metabolism, though we can gleam some insights from other fields. Transgenic murine experiments suggested that the reduced body weight of tankyrase 2 knockout mice was related to altered glucose uptake in adipose cells [14]. It was later shown however that these transgenic tankyrase 2 knockout mice exhibited an increase in ATP expenditure, fatty acid oxidation, and insulin-stimulated glucose consumption. This metabolic divergence all occurred without any effect on mitochondrial respiration [15]. Although the mechanism of this observed metabolic divergence remains opaque, others have elucidated some role of tankyrases in glucose metabolism. Tankyrases are capable of localizing not only to the nucleus, but the Golgi as well, where tankyrases interact with IRAP to promote membrane translocation of the glucose transporter GLUT4 [1]. Insulin-mediated exocytosis of GLUT4 storage vesicles requires both IRAP and tankyrase PARylation [1]. This tankyrase-mediated regulation of GLUT4 involves the WNT pathway protein AXIN [16]. Chemical inhibition of tankyrases by XAV929 have also shown augmented mitochondrial AXIN levels in HeLa cells as well as increased glycolysis absent of modified mitochondrial membrane potential [1]. Metabolic alteration in human naïve ESCs reveal a positive feedback loop on WNT signaling. NNMT knockout mutation in human naïve ESCs shows not only increased levels of H3K27Me3 repressive histone marks, but also contributed to inhibiting WNT signaling [12]. Thus, sustained WNT signaling is vital for maintaining the naïve pluripotent state via metabolic regulation in human ESCs. In XAV939 regulated pluripotent states it has been proposed that the manipulation of NAD+ flux and nicotinamide metabolism may alter PARP activities, including tankyrase-mediated WNT regulation [1]. Even still, the beforementioned studies paint a clear image that cellular metabolism and PARP activity are significantly intertwined via NAD+.