Could mammalian inorganic polyphosphate be a crucial signaling molecule in neurological disorders?

Since the early stages of life on earth, cellular metabolism has evolved to adapt to fluctuations in nutrient and oxygen availability. In this context, mammals, which are probably the organisms that show one of the highest levels of metabolic complexity, have developed an elegant system that uses constant and rechargeable energy sources of modulate their metabolism. This homeostasis is especially important in the central nervous system, as neurons and other cells in the brain are highly susceptible to fluctuations in nutrients and oxygen availability. At the molecular level, these energy sources are based on molecules that contain highly energetic bonds. The main metabolite with energy-rich bonds in mammalian cells is adenosine triphosphate. However, other molecules also present dynamic roles in cellular metabolism in these organisms. One of the lesser known of these molecules is inorganic polyphosphate (polyP). PolyP is an ancient polymer, which has been well conserved throughout evolution; it is present in every tissue of all studied organisms. PolyP bonds are isoenergetic to those found in adenosine triphosphate, and its role as a key energy metabolite has already been demonstrated by us and others in various systems, including mammalian cells (Guitart-Mampel et al., 2022). PolyP is ubiquitously distributed in the cell, although one of its preferred locations in mammals is mitochondria, where the vast majority of adenosine triphosphate is produced via oxidative phosphorylation. In fact, the levels of mitochondrial polyP seem to be intimately related to the status of the electron transfer chain. Moreover, the regulatory effects of polyP on some crucial mammalian mitochondrial processes that are closely related to the bioenergetic status of cells and usually deleteriously affected in neurological disorders; such as calcium and protein homeostasis, and the maintenance of the oxidative status; have also been described. Most likely as a consequence of its role in bioenergetics, polyP has also been reported to be involved in the cellular stress response of various organisms. For example, it has been shown that polyP can act as a primordial chaperone (Gray et al., 2014). This stress response is also often activated in neurological disorders. In fact, a recent and elegant study proposed that assessing the levels of polyP released by astrocytes could serve as a promising biomarker in amyotrophic lateral sclerosis and frontotemporal dementia (Arredondo et al., 2022). However, the molecular mechanism by which polyP exerts its effects on mitochondrial and cellular physiology, including bioenergetics, remains poorly understood, especially in mammals. One plausible explanation for these effects could be via the signaling role that polyP has shown in different organisms, including mammalian cells.

Much of the molecular pathways that are needed for the maintenance of proper mammalian cellular physiology in the central nervous system are sensitive to fluctuations in nutrient and oxygen levels. These fluctuations are intimately linked to the bioenergetics status of the cell. In fact, different metabolic pathways can detect the intracellular levels of energetic substrates and regulate specific cellular signaling nodes, which are different depending on the specific levels that are detected. The nutrient and energy sensing mechanism in mammalian cells is mainly composed of two central molecules: the mammalian target to rapamycin (mTOR), and the AMP-activated protein kinase (AMPK). Dysregulation of this sensing system has been broadly described in neurological disorders. Both mTOR and AMPK are regulated by phosphorylation and dephosphorylation events, which are mostly carried out by kinases. The availability of inorganic phosphate, which could be easily stored in polyP, is crucial for the regulatory activity of kinases in the mammalian metabolism. Therefore, the intracellular levels of polyP could play a critical role in the regulation of these sensing mechanisms, maybe just by acting as phosphate buffering molecules. In fact, preliminary data from our laboratory suggest a potent regulatory role for mitochondrial polyP in AMPK, especially under stress conditions, such as neurological disorders. Furthermore, the ability of polyP to polyphosphorylate proteins has already been demonstrated. Specifically, polyP chains can covalently attach to lysine residues of target proteins in a non-enzymatic way (Azevedo et al., 2015). This was first demonstrated in yeast, where the authors showed that polyphosphorylation affects the localization and physical interaction of two proteins: Nrs1 and Top1. To expand the understanding of lysine polyphosphorylation, other authors identified 15 protein targets, after screening for polyphosphorylation sites in yeast (Bentley-DeSousa et al., 2018). Many of these targets are protein sequences that are conserved in human cells. In fact, the authors showed that six of these targets are potential sites for polyphosphorylation in mammalian cells. While these results show a potent direct role of polyP in protein post-translational modifications, further studies should be conducted to empirically demonstrate this role in mammalian cells.

In the bibliography, we can find several studies that show the regulatory effects of polyP on mTOR. For example, using vascular endothelial cells (which are also present in the central nervous system), it has been demonstrated that polyP is involved in the regulation of mTOR complexes 1 and 2 (mTORC1 and mTORC2, respectively), by inducing the phosphorylation of p70S6K, via an AKT-dependent, but ERK-independent mechanism (Hassanian et al., 2015). Additionally, in a study conducted in mammalian endothelial cells, polyP induced the activation of the inflammatory response through binding to the receptor for advanced glycation end products (RAGE), and the purinergic receptor (P2YI). This binding stimulated the phosphorylation of the tuberous sclerosis complex, which resulted in the inhibition of the tuberous sclerosis complex, and leads to the activation of the mTOR pathway signaling (Dinarvand et al., 2014). The authors of this study also showed that the interaction of polyP with RAGE and P2YI activates mTORC1, once again, via an AKT-dependent mechanism. Moreover, during the pro-inflammatory signaling response in the vascular system, it has been demonstrated that activated platelets can secrete polyP. Considering this, mTORC could be activated via the platelets-secreted polyP, which could be further involved in the physiological pro-inflammatory signaling pathway described in vascular endothelial cells (Morrissey et al., 2012). The activation of inflammatory pathways, and more specifically of neuroinflammation, has been broadly demonstrated in many neurological disorders, including ischemic stroke. Besides its pro-inflammatory signaling role, the interaction between polyP and mTOR has also been reported to be involved in the modulation of cellular growth and proliferation. For example, a study conducted in mammary cancer cells showed that polyP regulates the in vitro activity of mTOR by its autophosphorylation, as well as that polyP increased the in vivo phosphorylation of one of the mTOR downstream targets, PHAS-I (Wang et al., 2003). The phosphorylation of PHAS-I by mTOR ultimately upregulates the expression of proteins involved in cell proliferation.

PolyP could also be involved in the regulation of the eukaryotic inositol polyphosphate multikinase (IPMK), which is a key enzyme in the formation of inositol polyP. Inositol polyP is an important regulator of neural physiology. Specifically, IPMK is the principal physiological generator of inositol pentakisphosphate (IP5, a molecule composed of a central ring of inositol and five lineal chains of different lengths of Pi, often polyP). Formation of IP5 is the rate-limiting step in the synthesis of inositol pyrophosphates, a group of molecules that often contain chains of polyP; and polyP could be a substrate for IPMK. The relationship between IP5 and polyP in the regulation of phosphate homeostasis in lower eukaryotes has already been suggested (Laha et al., 2021). This could indicate a potential role for polyP as a regulator of IPMK activity, which could further affect the regulatory signaling pathways in which this enzyme is involved. For example, IPMK mediates the amino acid signaling to mTORC1 (Kim et al., 2011). Interestingly, dysregulated IPMK affects not only energy homeostasis, but also the epigenetic regulation of gene transcription, which is also intimately related to the mechanisms involved in cell signaling. Further, IPMK is an important physiological regulator of AMPK, via control of glucose signaling. Therefore, by the modulation of the activity of IPMK, polyP could also indirectly affect the status of both mTOR and AMPK.

Calcium signaling is another crucial regulator of AMPK in mammalian cells, which is especially crucial in the central nervous system. In fact, the levels of calcium, especially those intra-mitochondrial, are dynamic, and closely related to the bioenergetics status of the cell, and its dysregulation has been reported in many neurological disorders, including ischemic stroke. Our group, using mammalian cells enzymatically depleted of polyP, showed the potent regulatory role exerted by polyP on calcium homeostasis (Solesio et al., 2020). Specifically, we demonstrated that polyP contributes to the effective and reversible buffering of calcium within mitochondria. Our findings were corroborated and expanded on by other groups. For example, using chondrocytes, it has been demonstrated that polyP can regulate anabolism as a consequence of its effects on calcium signaling (Gawri et al., 2022). Moreover, the role of polyP in the regulation of calcium influx was reported to be involved in signal transduction within the mammalian central nervous system. Specifically, polyP restricts the glutamate-induced calcium signal. In the presence of high glutamate concentrations, the ability of polyP to reduce the calcium influx was mediated via stimulation of P2YI receptors. The direct stimulation of P2YI receptors by polyP causes the release of neuromodulators, which will ultimately reduce the conductance of glutamate receptors and decrease drastically the calcium influx to cells (Maiolino et al., 2019). Finally, the role of polyP in the regulation of the generation of reactive oxygen species, which are well-known second messengers involved in cell signaling and whose dysregulation is also involved in all the most common neurological disorders, has also been demonstrated. For example, our research team showed that the enzymatic depletion of mitochondrial polyP in mammalian cells increases the generation of reactive oxygen species, most likely as a consequence of the deleterious effects exerted in oxidative phosphorylation by the lack of mitochondrial polyP (Hambardikar et al., 2022). All these signaling pathways in which polyP is involved in and that have relevance in the central nervous system are synthesized in Figure 1.

Figure 1.

Schematic representation of the regulatory effects of polyP in eukaryotic cell signaling.

These regulatory effects affect some of the main hallmarks which have been identified in the onset and progression of neurological disorders. Created with BioRender.com, using an institutional license (Rutgers University). AMPK: AMP-activated protein kinase; IPMK: inositol polyphosphate multikinase; mTOR: mammalian target of rapamycin; PTMs: post-translational modifications; ROS: reactive oxygen species.

All these data show the potent regulatory effects of polyP in mammalian cell signaling, including in processes, which are intimately related to the onset and progression of neurological disorders. While the results are clear, more research should be conducted in this field to further understand the mechanism(s) that underlies the interactions between polyP and the main signaling pathways. This mechanism(s) seems to be multifaceted. Signaling dysregulation, as a consequence of unbalanced bioenergetics, is present in many human pathologies. Therefore, increasing the knowledge of the effects of polyP on cell signaling could place the metabolism of polyP as a potent and innovative target in these conditions, as it was already suggested in the case of neurodegenerative disorders (Borden et al., 2021).

We kindly thank Mitch Maleki, Esq., for editing this manuscript. We apologize to colleagues whose work has not been cited due to the strict restrictions on the number of citations that are permitted.

RTDC postdoctoral fellowship is defrayed by an AHA Supplement to Promote Diversity in Science. MES is funded by AHA (Career Development Award), Rutgers University (StartUp Funds), and NIH (R00AG055701).