Human Prune Regulates the Metabolism of Mammalian Inorganic Polyphosphate and Bioenergetics.

Inorganic polyphosphate (polyP) is an evolutionarily conserved and ubiquitous polymer that is present in all studied organisms. PolyP consists of orthophosphates (Pi) linked together by phosphoanhydride bonds. The metabolism of polyP still remains poorly understood in higher eukaryotes. Currently, only F0F1-ATP synthase, Nudt3, and Prune have been proposed to be involved in this metabolism, although their exact roles and regulation in the context of polyP biology have not been fully elucidated. In the case of Prune, in vitro studies have shown that it exhibits exopolyphosphatase activity on very short-chain polyP (up to four units of Pi), in addition to its known cAMP phosphodiesterase (PDE) activity. Here, we expand upon studies regarding the effects of human Prune (h-Prune) on polyP metabolism. Our data show that recombinant h-Prune is unable to hydrolyze short (13-33 Pi) and medium (45-160 Pi) chains of polyP, which are the most common chain lengths of the polymer in mammalian cells. Moreover, we found that the knockdown of h-Prune (h-Prune KD) results in significantly decreased levels of polyP in HEK293 cells. Likewise, a reduction in the levels of polyP is also observed in Drosophila melanogaster loss-of-function mutants of the h-Prune ortholog. Furthermore, while the activity of ATP synthase, and the levels of ATP, are decreased in h-Prune KD HEK293 cells, the expression of ATP5A, which is a main component of the catalytic subunit of ATP synthase, is upregulated in the same cells, likely as a compensatory mechanism. Our results also show that the effects of h-Prune on mitochondrial bioenergetics are not a result of a loss of mitochondrial membrane potential or of significant changes in mitochondrial biomass. Overall, our work corroborates the role of polyP in mitochondrial bioenergetics. It also demonstrates a conserved effect of h-Prune on the metabolism of short- and medium-chain polyP (which are the predominant chain lengths found in mammalian cells). The effects of Prune in polyP are most likely exerted via the regulation of the activity of ATP synthase. Our findings pave the way for modifying the levels of polyP in mammalian cells, which could have pharmacological implications in many diseases where dysregulated bioenergetics has been demonstrated.

Cannabidiol repairs behavioral and brain disturbances in a model of fetal alcohol spectrum disorder.

Fetal alcohol spectrum disorder (FASD) includes neuropsychiatric disturbances related to gestational and lactational ethanol exposure. Available treatments are minimal and do not modulate ethanol-induced damage. Developing animal models simulating FASD is essential for understanding the underlying brain alterations and searching for efficient therapeutic approaches. The main goal of this study was to evaluate the effects of early and chronic cannabidiol (CBD) administration on offspring exposed to an animal model of FASD. Ethanol gavage (3 g/kg/12 h, p.o.) was administered to C57BL/6 J female mice, with a previous history of alcohol consumption, between gestational day 7 and postnatal day 21. On the weaning day, pups were separated by sex, and CBD administration began (30 mg/kg/day, i.p.). After 4-6 weeks of treatment, behavioral and neurobiological changes were analyzed. Mice exposed to the animal model of FASD showed higher anxiogenic and depressive-like behaviors and cognitive impairment that were evaluated through several experimental tests. These behaviors were accompanied by alterations in the gene, cellular and metabolomic targets. CBD administration normalized FASD model-induced emotional and cognitive disturbances, gene expression, and cellular changes with sex-dependent differences. CBD modulates the metabolomic changes detected in the hippocampus and prefrontal cortex. Interestingly, no changes were found in mitochondria or the oxidative status of the cells. These results suggest that the early and repeated administration of CBD modulated the long-lasting behavioral, gene and protein alterations induced by the FASD model, encouraging the possibility of performing clinical trials to evaluate the effects of CBD in children affected with FASD.

Toolkit for cellular studies of mammalian mitochondrial inorganic polyphosphate.

Introduction: Inorganic polyphosphate (polyP) is an ancient polymer which is extremely well-conserved throughout evolution, and found in every studied organism. PolyP is composed of orthophosphates linked together by high-energy bonds, similar to those found in ATP. The metabolism and the functions of polyP in prokaryotes and simple eukaryotes are well understood. However, little is known about its physiological roles in mammalian cells, mostly due to its unknown metabolism and lack of systematic methods and effective models for the study of polyP in these organisms. Methods: Here, we present a comprehensive set of genetically modified cellular models to study mammalian polyP. Specifically, we focus our studies on mitochondrial polyP, as previous studies have shown the potent regulatory role of mammalian polyP in the organelle, including bioenergetics, via mechanisms that are not yet fully understood. Results: Using SH-SY5Y cells, our results show that the enzymatic depletion of mitochondrial polyP affects the expression of genes involved in the maintenance of mitochondrial physiology, as well as the structure of the organelle. Furthermore, this depletion has deleterious effects on mitochondrial respiration, an effect that is dependent on the length of polyP. Our results also show that the depletion of mammalian polyP in other subcellular locations induces significant changes in gene expression and bioenergetics; as well as that SH-SY5Y cells are not viable when the amount and/or the length of polyP are increased in mitochondria. Discussion: Our findings expand on the crucial role of polyP in mammalian mitochondrial physiology and place our cell lines as a valid model to increase our knowledge of both mammalian polyP and mitochondrial physiology.

The Glacier Ice Worm, Mesenchytraeus solifugus, Elevates Mitochondrial Inorganic Polyphosphate (PolyP) Levels in Response to Stress.

The inorganic polymer, polyphosphate (polyP), is present in all organisms examined to date with putative functions ranging from the maintenance of bioenergetics to stress resilience and protein homeostasis. Bioenergetics in the glacier-obligate, segmented worm, Mesenchytraeus solifugus, is characterized by a paradoxical increase in intracellular ATP levels as temperatures decline. We show here that steady-state, mitochondrial polyP levels vary among species of Annelida, but were elevated only in M. solifugus in response to thermal stress. In contrast, polyP levels decreased with temperature in the mesophilic worm, Enchytraeus crypticus. These results identify fundamentally different bioenergetic strategies between closely related annelid worms, and suggest that I worm mitochondria maintain ATP and polyP in a dynamic equilibrium.

Enzymatic Depletion of Mitochondrial Inorganic Polyphosphate (polyP) Increases the Generation of Reactive Oxygen Species (ROS) and the Activity of the Pentose Phosphate Pathway (PPP) in Mammalian Cells.

Inorganic polyphosphate (polyP) is an ancient biopolymer that is well preserved throughout evolution and present in all studied organisms. In mammals, it shows a high co-localization with mitochondria, and it has been demonstrated to be involved in the homeostasis of key processes within the organelle, including mitochondrial bioenergetics. However, the exact extent of the effects of polyP on the regulation of cellular bioenergetics, as well as the mechanisms explaining these effects, still remain poorly understood. Here, using HEK293 mammalian cells under Wild-type (Wt) and MitoPPX (cells enzymatically depleted of mitochondrial polyP) conditions, we show that depletion of polyP within mitochondria increased oxidative stress conditions. This is characterized by enhanced mitochondrial O2- and intracellular H2O2 levels, which may be a consequence of the dysregulation of oxidative phosphorylation (OXPHOS) that we have demonstrated in MitoPPX cells in our previous work. These findings were associated with an increase in basal peroxiredoxin-1 (Prx1), superoxide dismutase-2 (SOD2), and thioredoxin (Trx) antioxidant protein levels. Using 13C-NMR and immunoblotting, we assayed the status of glycolysis and the pentose phosphate pathway (PPP) in Wt and MitoPPX cells. Our results show that MitoPPX cells display a significant increase in the activity of the PPP and an increase in the protein levels of transaldolase (TAL), which is a crucial component of the non-oxidative phase of the PPP and is involved in the regulation of oxidative stress. In addition, we observed a trend towards increased glycolysis in MitoPPX cells, which corroborates our prior work. Here, for the first time, we show the crucial role played by mitochondrial polyP in the regulation of mammalian redox homeostasis. Moreover, we demonstrate a significant effect of mitochondrial polyP on the regulation of global cellular bioenergetics in these cells.

Mitochondrial Inorganic Polyphosphate (polyP) Is a Potent Regulator of Mammalian Bioenergetics in SH-SY5Y Cells: A Proteomics and Metabolomics Study.

Inorganic polyphosphate (polyP) is an ancient, ubiquitous, and well-conserved polymer which is present in all the studied organisms. It is formed by individual subunits of orthophosphate which are linked by structurally similar bonds and isoenergetic to those found in ATP. While the metabolism and the physiological roles of polyP have already been described in some organisms, including bacteria and yeast, the exact role of this polymer in mammalian physiology still remains poorly understood. In these organisms, polyP shows a co-localization with mitochondria, and its role as a key regulator of the stress responses, including the maintenance of appropriate bioenergetics, has already been demonstrated by our group and others. Here, using Wild-type (Wt) and MitoPPX (cells enzymatically depleted of mitochondrial polyP) SH-SY5Y cells, we have conducted a comprehensive study of the status of cellular physiology, using proteomics and metabolomics approaches. Our results suggest a clear dysregulation of mitochondrial physiology, especially of bioenergetics, in MitoPPX cells when compared with Wt cells. Moreover, the effects induced by the enzymatic depletion of polyP are similar to those present in the mitochondrial dysfunction that is observed in neurodegenerative disorders and in neuronal aging. Based on our findings, the metabolism of mitochondrial polyP could be a valid and innovative pharmacological target in these conditions.

Is there a link between inorganic polyphosphate (polyP), mitochondria, and neurodegeneration?

Mitochondrial dysfunction - including increased apoptosis, calcium and protein dyshomeostasis within the organelle, and dysfunctional bioenergetics and oxidative status - is a common, early feature in all the major neurodegenerative diseases, including Alzheimer's Disease (AD) and Parkinson's Disease (PD). However, the exact molecular mechanisms that drive the organelle to dysfunction and ultimately to failure in these conditions are still not well described. Different authors have shown that inorganic polyphosphate (polyP), an ancient and well-conserved molecule, plays a key role in the regulation of mitochondrial physiology under basal conditions. PolyP, which is present in all studied organisms, is composed of chains of orthophosphates linked together by highly energetic phosphoanhydride bonds, similar to those found in ATP. This polymer shows a ubiquitous distribution, even if a high co-localization with mitochondria has been reported. It has been proposed that polyP might be an alternative to ATP for cellular energy storage in different organisms, as well as the implication of polyP in the regulation of many of the mitochondrial processes affected in AD and PD, including protein and calcium homeostasis. Here, we conduct a comprehensive review and discussion of the bibliography available regarding the role of polyP in the mitochondrial dysfunction present in AD and PD. Taking into account the data presented in this review, we postulate that polyP could be a valid, innovative and, plausible pharmacological target against mitochondrial dysfunction in AD and PD. However, further research should be conducted to better understand the exact role of polyP in neurodegeneration, as well as the metabolism of the polymer, and the effect of different lengths of polyP on cellular and mitochondrial physiology.