Fructose 1,6-bisphosphatase 2 (FBP2) is a multifunctional protein whose cellular functions depend on its oligomeric state. Forced FBP2 tetramerization has been linked to microtubule disruption and impaired mitochondrial trafficking, accompanied by abnormal mitochondrial morphology. Here, we identify MIC60 (mitofilin), a core element of the mitochondrial contact site and cristae organizing system (MICOS), as a potential mediator of these effects. Using proximity ligation assay, protein crosslinking combined with mass spectrometry, and ultrastructural analysis, we demonstrate that FBP2 is in close proximity to MIC60 under basal conditions and this proximity is reduced upon FBP2 tetramerization or partial FBP2 depletion. Loss of this proximity coincides with marked remodeling of inner-membrane ultrastructure. These findings are consistent with a working model in which dimeric FBP2 contributes to the coordination of microtubule-dependent mitochondrial positioning with MICOS-linked intramitochondrial organization, providing a plausible mechanistic bridge between metabolic cues (AMP/NAD+) and mitochondrial structural integrity.

Introduction and Objective: Diabetes is characterized by chronic hyperglycemia and damage to the central nervous system, which may lead to impaired cognitive function. Despite the linear correlation between brain and blood glucose concentrations, most neurophysiological studies use supraphysiological glucose levels (≥25 mM). Such conditions do not reflect normal neuronal function and correspond to extreme hyperglycemia. Here, we examined how mitochondrial metabolic responses to long-term potentiation (LTP), a fundamental cellular mechanism of learning and memory formation, depend on glucose availability. Methods: Primary hippocampal neurons were cultured for 14 days under either in physiological (2.5 mM) or hyperglycemic (25 mM) brain glucose conditions. On day 14, LTP was chemically induced. Cells and media were collected for metabolomic profiling using gas chromatography mass spectrometry. Results: Neuronal viability was higher under physiological glucose compared to hyperglycemia. At baseline, neurons cultured at 2.5 mM glucose exhibited reduced pyruvate, lactate, alanine and neurotransmitters while maintaining TCA cycle intermediates. Upon LTP induction, metabolic responses differed between glucose conditions, with accumulation of TCA intermediates and increased lactate release at 2.5 mM glucose, contrasted by depletion of mitochondrial metabolites and reduced lactate secretion at 25 mM glucose. Importantly, LTP induction was preserved under physiological glucose as evidenced by immunofluorescence measurements of c-Fos expression. Conclusion: Glucose availability changes neuronal metabolic responses without impairing LTP induction. These results show that studying synaptic plasticity under supraphysiological glucose conditions, such as hyperglycemia, may be misleading because neuronal metabolism differs substantially, highlighting the need for physiologically relevant models when investigating diabetes-related cognitive impairment. Disclosure N. Pudelko-Malik: None. D. Drulis-Fajdasz: None. M.M. Fydryszewski: None. S.C. Burgess: None. P. Mlynarz: None. D. Rakus: None. S. Deja: None. Funding The Kosciuszko Foundation the American Centre of Polish Culture, National Institutes of Health (K01DK133630), Polish National Science Centre (2020/37/B/NZ4/00808)

Cells modulate their physiology through multiple mechanisms-cell-cell contacts and autocrine/paracrine signaling, including via extracellular vesicles (EVs). In this study, we exposed mouse hippocampal astrocyte and neuron monocultures to EVs from the opposing cell type and subsequently performed RNA sequencing to examine transcriptomic changes. Mass spectrometry was used to analyze the proteomes of EVs from astrocyte and neuron monocultures, as well as from astrocyte-neuron co-cultures, to investigate the molecular basis of EVs-induced transcriptomic alterations and to determine the extent to which cells adjust EV cargo in response to feedback signals. EVs secreted by both cell types induced cell-specific transcriptomic changes in target cells, related to migration, proliferation, differentiation, and energy production. Unique changes in the proteome of EVs from astrocytic-neuronal co-cultures highlighted the dynamic regulation of signaling molecule secretion via cell interactions.

Despite their widespread use as a research model, a comprehensive, quantitative proteomic profile of the cultured hippocampal neurons has remained unexplored. Here, we provide the first global proteomic characterization of primary murine hippocampal neurons cultured for 14 days under near-physiological glucose conditions (2.5 mM). Using label-free mass spectrometry and the Total Protein Approach (TPA), we quantified over 5,500 proteins, revealing a metabolic signature consistent with oxidative phosphorylation and a neuron-specific proteome largely devoid of astrocytic markers. Unexpectedly, glial fibrillary acidic protein (GFAP) was highly abundant in pure neuronal cultures, and its knockdown impaired neuronal viability and morphology. These findings suggest a compensatory role for GFAP in neurons cultured without astrocytic support. Additionally, our data highlight significant underexpression of key synaptic proteins compared to hippocampal tissue, underscoring the limitations of neuron-only culture systems. This study establishes a new molecular reference for hippocampal neurons and offers insights into neuronal adaptation in simplified environments.

Pancreatic cancer is one of the most poorly prognosed types of cancer, with a low survival rate. Cancer cells exhibit altered and rapid metabolism, and in a process known as aerobic glycolysis, they metabolize glucose to lactate even in the presence of sufficient oxygen. Altered metabolism is a hallmark of cancer, making it a promising therapeutic target. There are many potential inhibitors of cancer cell metabolism, including auranofin and syrosingopine. Auranofin induces oxidative stress in cells by generating reactive oxygen species (ROS). Meanwhile, syrosingopine is an inhibitor of the two lactate transporters, MCT1 and MCT4, which contribute to intracellular acidification. Our studies demonstrated the high biological activity of the combination of auranofin and syrosingopine against pancreatic cancer in both in vitro and in vivo models. Our proposed mechanism of action for this drug combination is based on the simultaneous induction of oxidative stress and inhibition of lactate transporters, which consequently directs cancer cells to the apoptosis pathway. ### Competing Interest Statement The authors have declared no competing interest.

Glucose is a predominant fuel for the brain supporting its high energy demand associated with neuronal signaling and synaptic activity. Long-term potentiation (LTP) is required for learning and memory formation by generating long lasting increase in synaptic strength and signal transmission between two neurons. While the electrophysiological bases of LTP are well established, much less is known about the metabolic demands of neurons involved in LTP. Common protocols used to examine synaptic activity rely on high glucose concentrations which are far from physiological glucose levels found in the brain. Here we used primary hippocampal neurons cultured under physiological (2.5 mM) and high (25 mM) glucose to investigate the metabolic effects of chemically induced LTP. Physiological glucose was associated with neuronal survival while high glucose promoted "PAS granule" accumulation. Changes in glucose altered extracellular lactate and pyruvate concentrations and affected key intracellular metabolic intermediates and neurotransmitter levels in neuronal cells without depleting the TCA cycle. LTP induction was comparable, but mitochondrial and neurotransmitter response to LTP was differentially affected physiological and high glucose conditions. Glycogen phosphorylase inhibition had minimal effects in physiological glucose but impaired synaptic responses and altered metabolite dynamics in high glucose. Our findings demonstrate that neuronal mitochondrial metabolism is closely linked to synaptic plasticity and highlight the importance of studying neurophysiological activity physiologically relevant glucose conditions.

Inhibition of glycogen phosphorylase (Pyg) - a regulatory enzyme of glycogen phosphorolysis - influences memory formation in rodents. We have previously shown that 2-week intraperitoneal administration of a Pyg inhibitor BAY U6751 stimulated the "rejuvenation" of the hippocampal proteome and dendritic spines morphology and improved cognitive skills of old mice. Given the tedious nature of daily intraperitoneal drug administration, in this study we investigated whether a single dose of BAY U6751 could induce enduring behavioral effects. Obtained results support the efficacy of such treatment in significantly improving the cognitive performance of 20-22-month-old mice. Metabolomic analysis of alterations observed in the hippocampus, cerebellum, and cortex reveal that the inhibition of glycogen phosphorolysis impacts not only glucose metabolism but also various other metabolic processes.

Fructose 1,6-bisphosphatase 2 (Fbp2) is a regulatory enzyme of gluco- and glyconeogenesis which, in the course of evolution, acquired non-catalytic functions. Fbp2 promotes cell survival during calcium stress, regulates glycolysis via inhibition of Hif-1α activity, and is indispensable for the formation of long-term potentiation in hippocampus. In hippocampal astrocytes, the amount of Fbp2 protein is reduced by signals delivered in neuronal extracellular vesicles (NEVs) through an unknown mechanism. The physiological role of Fbp2 (determined by its subcellular localization/interactions) depends on its oligomeric state and thus, we asked whether the cargo of NEVs is sufficient to change also the ratio of Fbp2 dimer/tetramer and, consequently, influence astrocyte basal metabolism. We found that the NEVs cargo reduced the Fbp2 mRNA level, stimulated the enzyme degradation and affected the cellular titers of different oligomeric forms of Fbp2. This was accompanied with increased glucose uptake and lactate release by astrocytes. Our results revealed that neuronal signals delivered to astrocytes in NEVs provide the necessary balance between enzymatic and non-enzymatic functions of Fbp2, influencing not only its amount but also subcellular localization. This may allow for the metabolic adjustments and ensure protection of mitochondrial membrane potential during the neuronal activity-related increase in astrocytic [Ca2+].

Fructose 1,6-bisphosphatase (FBP) is a regulatory enzyme of gluconeogenesis that also influences in a non-catalytic manner - via protein-protein interactions - cell cycle-dependent events, mitochondria biogenesis and polarization, synaptic plasticity and even cancer progression. FBP reduces glycolytic capacity of cells via blocking HIF-1α transcriptional activity and modulating NF-κB action, and influences oxidative metabolism by binding to c-MYC. Because FBP limits the energy-producing potential of cells and because a reduction of FBP amounts is observed in cancer cells, FBP is considered to be an anti-oncogenic protein. This is supported by the observation that cancer cells overexpress aldolase A (ALDOA), a pro-oncogenic protein that can bind to FBP and potentially block its anti-oncogenic activity. Interestingly, only the muscle isozyme of FBP (FBP2) interacts strongly with ALDOA, whereas the binding of the liver isozyme (FBP1) to ALDOA is more than an order of magnitude weaker. Here, we briefly review the most important evidence supporting the anti-oncogenic function of FBP and discuss what structural properties of the two FBP isozymes allow FBP2, rather than FBP1, to exert more flexible anticancer functions.

Inhibition of glycogen breakdown blocks memory formation in young animals, but it stimulates the maintenance of the long-term potentiation, a cellular mechanism of memory formation, in hippocampal slices of old animals. Here, we report that a 2-week treatment with glycogen phosphorylase inhibitor BAY U6751 alleviated memory deficits and stimulated neuroplasticity in old mice. Using the 2-Novel Object Recognition and Novel Object Location tests, we discovered that the prolonged intraperitoneal administration of BAY U6751 improved memory formation in old mice. This was accompanied by changes in morphology of dendritic spines in hippocampal neurons, and by "rejuvenation" of hippocampal proteome. In contrast, in young animals, inhibition of glycogen degradation impaired memory formation; however, as in old mice, it did not alter significantly the morphology and density of cortical dendritic spines. Our findings provide evidence that prolonged inhibition of glycogen phosphorolysis improves memory formation of old animals. This could lead to the development of new strategies for treatment of age-related memory deficits.

Recently, we have shown that the physiological roles of a multifunctional protein fructose 1,6-bisphosphatase 2 (FBP2, also called muscle FBP) depend on the oligomeric state of the protein. Here, we present several lines of evidence that in HL-1 cardiomyocytes, a forced, chemically induced reduction in the FBP2 dimer-tetramer ratio that imitates AMP and NAD+ action and restricts FBP2-mitochondria interaction, results in an increase in Tau phosphorylation, augmentation of FBP2-Tau and FBP2-MAP1B interactions, disturbance of tubulin network, marked reduction in the speed of mitochondrial trafficking and increase in mitophagy. These results not only highlight the significance of oligomerization for the regulation of FBP2 physiological role in the cell, but they also demonstrate a novel, important cellular function of this multitasking protein—a function that might be crucial for processes that take place during physiological and pathological cardiac remodeling, and during the onset of diseases which are rooted in the destabilization of MT and/or mitochondrial network dynamics.

The TP53 tumor suppressor is mutated in ~75% of pancreatic cancers. The mutant TP53 protein in pancreatic ductal adenocarcinomas (PDAC) promotes tumor growth and metastasis. Attempts have been made to develop molecules that restore at least some of the properties of wild-type (WT) TP53. APR-246 is one such molecule, and it is referred to as a mutant TP53 reactivator. To understand the potential of APR-246 to sensitize PDAC cells to chemotherapy, we introduced a vector encoding WT-TP53 into two PDAC cell lines, one lacking the expression of TP53 (PANC-28) and one with a gain-of-function (GOF) mutant TP53 (MIA-PaCa-2). APR-246 increased drug sensitivity in the cells containing either a WT or mutant TP53 protein with GOF activity, but not in cells that lacked TP53. The introduction of WT-T53 into PANC-28 cells increased their sensitivity to the TP53 reactivator, chemotherapeutic drugs, and signal transduction inhibitors. The addition of WT-TP53 to PDAC cells with GOF TP53 also increased their sensitivity to the drugs and therapeutics, indicating that APR-246 could function in cells with WT-TP53 and GOF TP53. These results highlight the importance of knowledge of the type of TP53 mutation that is present in cancer patients before the administration of drugs which function through the reactivation of TP53.

Phosphoglycerate mutase (PGAM) is a glycolytic enzyme converting 3-phosphoglycerate to 2-phosphoglycerate, which in mammalian cells is expressed in two isoforms: brain (PGAM1) and muscle (PGAM2). Recently, it was shown that besides its enzymatic function, PGAM2 can be imported to the cell nucleus where it co-localizes with the nucleoli. It was suggested that it functions there to stabilize the nucleolar structure, maintain mRNA expression, and assist in the assembly of new pre-ribosomal subunits. However, the precise mechanism by which the protein translocates to the nucleus is unknown. In this study, we present the first crystal structure of PGAM2, identify the residues involved in the nuclear localization of the protein and propose that PGAM contains a “quaternary nuclear localization sequence (NLS)”, i.e., one that consists of residues from different protein chains. Additionally, we identify potential interaction partners for PGAM2 in the nucleoli and demonstrate that 14-3-3ζ/δ is indeed an interaction partner of PGAM2 in the nucleus. We also present evidence that the insulin/IGF1–PI3K–Akt–mTOR signaling pathway is responsible for the nuclear localization of PGAM2.

Muscle fructose-1,6-bisphosphatase (FBPase), which catalyzes the hydrolysis of fructose-1,6-bisphosphate (F1,6BP) to fructose-6-phosphate (F6P) and inorganic phosphate, regulates glucose homeostasis by controlling the glyconeogenic pathway. FBPase requires divalent cations, such as Mg2+, Mn2+, or Zn2+, for its catalytic activity; however, calcium ions inhibit the muscle isoform of FBPase by interrupting the movement of the catalytic loop. It has been shown that residue E69 in this loop plays a key role in the sensitivity of muscle FBPase towards calcium ions. The study presented here is based on five crystal structures of wild-type human muscle FBPase and its E69Q mutant in complexes with the substrate and product of the enzymatic reaction, namely F1,6BP and F6P. The ligands are bound in the active site of the studied proteins in the same manner and have excellent definition in the electron density maps. In all studied crystals, the homotetrameric enzyme assumes the same cruciform quaternary structure, with the κ angle, which describes the orientation of the upper dimer with respect to the lower dimer, of -85o. This unusual quaternary arrangement of the subunits, characteristic of the R-state of muscle FBPase, is also observed in solution by small-angle X-ray scattering (SAXS).

Leukodystrophies are genetic disorders of cerebral white matter that almost exclusively have a progressive disease course. We became aware of three members of a family with a disorder characterized by a sudden loss of all previously acquired abilities around 1 year of age followed by almost complete recovery within 2 years. Cerebral MRI and myelin sensitive imaging showed a pronounced demyelination that progressed for several months despite signs of clinical improvement and was followed by remyelination. Exome sequencing did not-identify any mutations in known leukodystrophy genes but revealed a heterozygous variant in the FBP2 gene, c.343G>A, p. Val115Met, shared by the affected family members. Cerebral MRI of other family members demonstrated similar white matter abnormalities in all carriers of the variant in FBP2. The FBP2 gene codes for muscle fructose 1,6-bisphosphatase, an enzyme involved in gluconeogenesis that is highly expressed in brain tissue. Biochemical analysis showed that the variant has a dominant negative effect on enzymatic activity, substrate affinity, cooperativity and thermal stability. Moreover, it also affects the non-canonical functions of muscle fructose 1,6-bisphosphatase involved in mitochondrial protection and regulation of several nuclear processes. In patients' fibroblasts, muscle fructose 1,6-bisphosphatase shows no colocalization with mitochondria and nuclei leading to increased reactive oxygen species production and a disturbed mitochondrial network. In conclusion, the results of this study indicate that the variant in FBP2 disturbs cerebral energy metabolism and is associated with a novel remitting leukodystrophy.

Aging is associated with a general decline of cognitive functions, and it is widely accepted that this decline results from changes in the expression of proteins involved in regulation of synaptic plasticity. However, several lines of evidence have accumulated that suggest that the impaired function of the aged brain may be related to significant alterations in the energy metabolism. In the current study, we employed the label-free "Total protein approach" (TPA) method to focus on the similarities and differences in energy metabolism proteomes of young (1-month-old) and aged (22-month-old) murine brains. We quantified over 7000 proteins in each of the following three analyzed brain structures: the hippocampus, the cerebral cortex and the cerebellum. To the best of our knowledge, this is the most extensive quantitative proteomic description of energy metabolism pathways during the physiological aging of mice. The analysis demonstrates that aging does not significantly affect the abundance of total proteins in the studied brain structures, however, the levels of proteins constituting energy metabolism pathways differ significantly between young and aged mice.