Effect of intracerebroventricular administration of alglucosidase alfa in two mouse models of Lafora disease: Relevance for clinical practice.

Lafora disease is a rare and fatal form of progressive myoclonic epilepsy with onset during early adolescence. The disease is caused by mutations in EPM2A, encoding laforin, or EPM2B, encoding malin. Both proteins have functions that affect glycogen metabolism, including glycogen dephosphorylation by laforin and ubiquitination of enzymes involved in glycogen metabolism by malin. Lack of function of laforin or malin results in the accumulation of polyglucosan that forms Lafora bodies in the central nervous system and other tissues. Enzyme replacement therapy through intravenous administration of alglucosidase alfa (Myozyme®) has shown beneficial effects removing polyglucosan aggregates in Pompe disease. We evaluated the effectiveness of intracerebroventricular administration of alglucosidase alfa in the Epm2a-/- knock-out and Epm2aR240X knock-in mouse models of Lafora disease. Seven days after a single intracerebroventricular injection of alglucosidase alfa in 12-month-old Epm2a-/- and Epm2aR240X mice, the number of Lafora bodies was not reduced. Additionally, a prolonged infusion of alglucosidase alfa for 2 or 4 weeks in 6- and 9-month-old Epm2a-/- mice did not result in a reduction in the number of LBs or the amount of glycogen in the brain. These findings hold particular significance in guiding a rational approach to the utilization of novel therapies in Lafora disease.

Small-molecule inhibition of glycogen synthase 1 for the treatment of Pompe disease and other glycogen storage disorders.

Glycogen synthase 1 (GYS1), the rate-limiting enzyme in muscle glycogen synthesis, plays a central role in energy homeostasis and has been proposed as a therapeutic target in multiple glycogen storage diseases. Despite decades of investigation, there are no known potent, selective small-molecule inhibitors of this enzyme. Here, we report the preclinical characterization of MZ-101, a small molecule that potently inhibits GYS1 in vitro and in vivo without inhibiting GYS2, a related isoform essential for synthesizing liver glycogen. Chronic treatment with MZ-101 depleted muscle glycogen and was well tolerated in mice. Pompe disease, a glycogen storage disease caused by mutations in acid α glucosidase (GAA), results in pathological accumulation of glycogen and consequent autophagolysosomal abnormalities, metabolic dysregulation, and muscle atrophy. Enzyme replacement therapy (ERT) with recombinant GAA is the only approved treatment for Pompe disease, but it requires frequent infusions, and efficacy is limited by suboptimal skeletal muscle distribution. In a mouse model of Pompe disease, chronic oral administration of MZ-101 alone reduced glycogen buildup in skeletal muscle with comparable efficacy to ERT. In addition, treatment with MZ-101 in combination with ERT had an additive effect and could normalize muscle glycogen concentrations. Biochemical, metabolomic, and transcriptomic analyses of muscle tissue demonstrated that lowering of glycogen concentrations with MZ-101, alone or in combination with ERT, corrected the cellular pathology in this mouse model. These data suggest that substrate reduction therapy with GYS1 inhibition may be a promising therapeutic approach for Pompe disease and other glycogen storage diseases.

MetaVision3D: Automated Framework for the Generation of Spatial Metabolome Atlas in 3D.

High-resolution spatial imaging is transforming our understanding of foundational biology. Spatial metabolomics is an emerging field that enables the dissection of the complex metabolic landscape and heterogeneity from a thin tissue section. Currently, spatial metabolism highlights the remarkable complexity in two-dimensional space and is poised to be extended into the three-dimensional world of biology. Here, we introduce MetaVision3D, a novel pipeline driven by computer vision techniques for the transformation of serial 2D MALDI mass spectrometry imaging sections into a high-resolution 3D spatial metabolome. Our framework employs advanced algorithms for image registration, normalization, and interpolation to enable the integration of serial 2D tissue sections, thereby generating a comprehensive 3D model of unique diverse metabolites across host tissues at mesoscale. As a proof of principle, MetaVision3D was utilized to generate the mouse brain 3D metabolome atlas (available at https://metavision3d.rc.ufl.edu/ ) as an interactive online database and web server to further advance brain metabolism and related research.

RIT1 regulation of CNS lipids RIT1 deficiency Alters cerebral lipid metabolism and reduces white matter tract oligodendrocytes and conduction velocities.

Oligodendrocytes (OLs) generate lipid-rich myelin membranes that wrap axons to enable efficient transmission of electrical impulses. Using a RIT1 knockout mouse model and in situ high-resolution matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) coupled with MS-based lipidomic analysis to determine the contribution of RIT1 to lipid homeostasis. Here, we report that RIT1 loss is associated with altered lipid levels in the central nervous system (CNS), including myelin-associated lipids within the corpus callosum (CC). Perturbed lipid metabolism was correlated with reduced numbers of OLs, but increased numbers of GFAP+ glia, in the CC, but not in grey matter. This was accompanied by reduced myelin protein expression and axonal conduction deficits. Behavioral analyses revealed significant changes in voluntary locomotor activity and anxiety-like behavior in RIT1KO mice. Together, these data reveal an unexpected role for RIT1 in the regulation of cerebral lipid metabolism, which coincide with altered white matter tract oligodendrocyte levels, reduced axonal conduction velocity, and behavioral abnormalities in the CNS.

Inhibition of mitochondrial fission activates glycogen synthesis to support cell survival in colon cancer.

Metabolic reprogramming has been recognized as one of the major mechanisms that fuel tumor initiation and progression. Our previous studies demonstrate that activation of Drp1 promotes fatty acid oxidation and downstream Wnt signaling. Here we investigate the role of Drp1 in regulating glycogen metabolism in colon cancer. Knockdown of Drp1 decreases mitochondrial respiration without increasing glycolysis. Analysis of cellular metabolites reveals that the levels of glucose-6-phosphate, a precursor for glycogenesis, are significantly elevated whereas pyruvate and other TCA cycle metabolites remain unchanged in Drp1 knockdown cells. Additionally, silencing Drp1 activates AMPK to stimulate the expression glycogen synthase 1 (GYS1) mRNA and promote glycogen storage. Using 3D organoids from Apcf/f/Villin-CreERT2 models, we show that glycogen levels are elevated in tumor organoids upon genetic deletion of Drp1. Similarly, increased GYS1 expression and glycogen accumulation are detected in xenograft tumors derived from Drp1 knockdown colon cancer cells. Functionally, increased glycogen storage provides survival advantage to Drp1 knockdown cells. Co-targeting glycogen phosphorylase-mediated glycogenolysis sensitizes Drp1 knockdown cells to chemotherapy drug treatment. Taken together, our results suggest that Drp1-loss activates glucose uptake and glycogenesis as compensative metabolic pathways to promote cell survival. Combined inhibition of glycogen metabolism may enhance the efficacy of chemotherapeutic agents for colon cancer treatment.

TgLaforin, a glucan phosphatase, reveals the dynamic role of storage polysaccharides in Toxoplasma gondii tachyzoites and bradyzoites.

The asexual stages of Toxoplasma gondii are defined by the rapidly growing tachyzoite during the acute infection and by the slow growing bradyzoite housed within tissue cysts during the chronic infection. These stages represent unique physiological states, each with distinct glucans reflecting differing metabolic needs. A defining feature of T. gondii bradyzoites is the presence of insoluble storage glucans known as amylopectin granules (AGs) that are believed to play a role in reactivation, but their functions during the chronic infection remain largely unexplored. More recently, the presence of storage glucans has been recognized in tachyzoites where their precise function and architecture have yet to be fully defined. Importantly, the T. gondii genome encodes activities needed for glucan turnover: a glucan phosphatase (TgLaforin; TGME49_205290) and a glucan kinase (TgGWD; TGME49_214260) that catalyze a cycle of reversible glucan phosphorylation required for glucan degradation by amylases. The expression of these enzymes in tachyzoites supports the existence of a storage glucan, evidence that is corroborated by specific labeling with the anti-glycogen antibody IV58B6. Disruption of reversible glucan phosphorylation via a CRISPR/Cas9 knockout (KO) of TgLaforin revealed no growth defects under nutrient-replete conditions in tachyzoites. However, the growth of TgLaforin-KO tachyzoites was severely stunted when starved of glutamine, even under glucose replete conditions. The loss of TgLaforin also resulted in the attenuation of acute virulence in mice accompanied by a lower cyst burden. Defective cyst formation due to profound changes in AG morphology was also observed in TgLaforin-KO parasites, both in vitro and in vivo. Together, these data demonstrate the importance of glucan turnover across the T. gondii asexual cycle. These findings, alongside our previously identified class of small molecules that inhibit TgLaforin, implicate reversible glucan phosphorylation as a legitimate target for the development of new drugs against chronic T. gondii infections.

Gys1 Antisense Therapy Prevents Disease-Driving Aggregates and Epileptiform Discharges in a Lafora Disease Mouse Model.

Patients with Lafora disease have a mutation in EPM2A or EPM2B, resulting in dysregulation of glycogen metabolism throughout the body and aberrant glycogen molecules that aggregate into Lafora bodies. Lafora bodies are particularly damaging in the brain, where the aggregation drives seizures with increasing severity and frequency, coupled with neurodegeneration. Previous work employed mouse genetic models to reduce glycogen synthesis by approximately 50%, and this strategy significantly reduced Lafora body formation and disease phenotypes. Therefore, an antisense oligonucleotide (ASO) was developed to reduce glycogen synthesis in the brain by targeting glycogen synthase 1 (Gys1). To test the distribution and efficacy of this drug, the Gys1-ASO was administered to Epm2b-/- mice via intracerebroventricular administration at 4, 7, and 10 months. The mice were then sacrificed at 13 months and their brains analyzed for Gys1 expression, glycogen aggregation, and neuronal excitability. The mice treated with Gys1-ASO exhibited decreased Gys1 protein levels, decreased glycogen aggregation, and reduced epileptiform discharges compared to untreated Epm2b-/- mice. This work provides proof of concept that a Gys1-ASO halts disease progression of EPM2B mutations of Lafora disease.

Prognostic value of pathogenic variants in Lafora Disease: systematic review and meta-analysis of patient-level data.

BACKGROUND: Lafora disease (LD) is a fatal form of progressive myoclonic epilepsy caused by biallelic pathogenic variants in EPM2A or NHLRC1. With a few exceptions, the influence of genetic factors on disease progression has yet to be confirmed. We present a systematic review and meta-analysis of the known pathogenic variants to identify genotype-phenotype correlations. METHODS: We collected all reported cases with genetically-confirmed LD containing data on disease history. Pathogenic variants were classified into missense (MS) and protein-truncating (PT). Three genotype classes were defined according to the combination of the variants: MS/MS, MS/PT, and PT/PT. Time-to-event analysis was performed to evaluate survival and loss of autonomy. RESULTS: 250 cases described in 70 articles were included. The mutated gene was NHLRC1 in 56% and EPM2A in 44% of cases. 114 pathogenic variants (67 EPM2A; 47 NHLRC1) were identified. The NHLRC1 genotype PT/PT was associated with shorter survival [HR 2.88; 95% CI 1.23-6.78] and a trend of higher probability of loss of autonomy [HR 2.03, 95% CI 0.75-5.56] at the multivariable Cox regression analysis. The population carrying the homozygous p.Asp146Asn variant of NHLRC1 genotype was confirmed to have a more favourable prognosis in terms of disease duration. CONCLUSIONS: This study demonstrates the existence of prognostic genetic factors in LD, namely the genotype defined according to the functional impact of the pathogenic variants. Although the reasons why NHLRC1 genotype PT/PT is associated with a poorer prognosis have yet to be fully elucidated, it may be speculated that malin plays a pivotal role in LD pathogenesis.

Spatial Metabolome Lipidome and Glycome from a Single brain Section.

Metabolites, lipids, and glycans are fundamental biomolecules involved in complex biological systems. They are metabolically channeled through a myriad of pathways and molecular processes that define the physiology and pathology of an organism. Here, we present a blueprint for the simultaneous analysis of spatial metabolome, lipidome, and glycome from a single tissue section using mass spectrometry imaging. Complimenting an original experimental protocol, our workflow includes a computational framework called Spatial Augmented Multiomics Interface (Sami) that offers multiomics integration, high dimensionality clustering, spatial anatomical mapping with matched multiomics features, and metabolic pathway enrichment to providing unprecedented insights into the spatial distribution and interaction of these biomolecules in mammalian tissue biology.

Current avenues of gene therapy in Pompe disease.

PURPOSE OF REVIEW: Pompe disease is a rare, inherited, devastating condition that causes progressive weakness, cardiomyopathy and neuromotor disease due to the accumulation of glycogen in striated and smooth muscle, as well as neurons. While enzyme replacement therapy has dramatically changed the outcome of patients with the disease, this strategy has several limitations. Gene therapy in Pompe disease constitutes an attractive approach due to the multisystem aspects of the disease and need to address the central nervous system manifestations. This review highlights the recent work in this field, including methods, progress, shortcomings, and future directions. RECENT FINDINGS: Recombinant adeno-associated virus (rAAV) and lentiviral vectors (LV) are well studied platforms for gene therapy in Pompe disease. These products can be further adapted for safe and efficient administration with concomitant immunosuppression, with the modification of specific receptors or codon optimization. rAAV has been studied in multiple clinical trials demonstrating safety and tolerability. SUMMARY: Gene therapy for the treatment of patients with Pompe disease is feasible and offers an opportunity to fully correct the principal pathology leading to cellular glycogen accumulation. Further work is needed to overcome the limitations related to vector production, immunologic reactions and redosing.

The multifaceted roles of the brain glycogen.

Glycogen is a biologically essential macromolecule that is directly involved in multiple human diseases. While its primary role in carbohydrate storage and energy metabolism in the liver and muscle is well characterized, recent research has highlighted critical metabolic and non-metabolic roles for glycogen in the brain. In this review, the emerging roles of glycogen homeostasis in the healthy and diseased brain are discussed with a focus on advancing our understanding of the role of glycogen in the brain. Innovative technologies that have led to novel insights into glycogen functions are detailed. Key insights into how cellular localization impacts neuronal and glial function are discussed. Perturbed glycogen functions are observed in multiple disorders of the brain, including where it serves as a disease driver in the emerging category of neurological glycogen storage diseases (n-GSDs). n-GSDs include Lafora disease (LD), adult polyglucosan body disease (APBD), Cori disease, Glucose transporter type 1 deficiency syndrome (G1D), GSD0b, and late-onset Pompe disease (PD). They are neurogenetic disorders characterized by aberrant glycogen which results in devastating neurological and systemic symptoms. In the most severe cases, rapid neurodegeneration coupled with dementia results in death soon after diagnosis. Finally, we discuss current treatment strategies that are currently being developed and have the potential to be of great benefit to patients with n-GSD. Taken together, novel technologies and biological insights have resulted in a renaissance in brain glycogen that dramatically advanced our understanding of both biology and disease. Future studies are needed to expand our understanding and the multifaceted roles of glycogen and effectively apply these insights to human disease.

Glycogen accumulation modulates life span in a mouse model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease characterized by the progressive loss of motor neurons in the spinal cord. Glial cells, including astrocytes and microglia, have been shown to contribute to neurodegeneration in ALS, and metabolic dysfunction plays an important role in the progression of the disease. Glycogen is a soluble polymer of glucose found at low levels in the central nervous system that plays an important role in memory formation, synaptic plasticity, and the prevention of seizures. However, its accumulation in astrocytes and/or neurons is associated with pathological conditions and aging. Importantly, glycogen accumulation has been reported in the spinal cord of human ALS patients and mouse models. In the present work, using the SOD1G93A mouse model of ALS, we show that glycogen accumulates in the spinal cord and brainstem during symptomatic and end stages of the disease and that the accumulated glycogen is associated with reactive astrocytes. To study the contribution of glycogen to ALS progression, we generated SOD1G93A mice with reduced glycogen synthesis (SOD1G93A GShet mice). SOD1G93A GShet mice had a significantly longer life span than SOD1G93A mice and showed lower levels of the astrocytic pro-inflammatory cytokine Cxcl10, suggesting that the accumulation of glycogen is associated with an inflammatory response. Supporting this, inducing an increase in glycogen synthesis reduced life span in SOD1G93A mice. Altogether, these results suggest that glycogen in reactive astrocytes contributes to neurotoxicity and disease progression in ALS.

In situ microwave fixation provides an instantaneous snapshot of the brain metabolome.

Brain glucose metabolism is highly heterogeneous among brain regions and continues postmortem. In particular, we demonstrate exhaustion of glycogen and glucose and an increase in lactate production during conventional rapid brain resection and preservation by liquid nitrogen. In contrast, we show that these postmortem changes are not observed with simultaneous animal sacrifice and in situ fixation with focused, high-power microwave. We further employ microwave fixation to define brain glucose metabolism in the mouse model of streptozotocin-induced type 1 diabetes. Using both total pool and isotope tracing analyses, we identified global glucose hypometabolism in multiple brain regions, evidenced by reduced 13C enrichment into glycogen, glycolysis, and the tricarboxylic acid (TCA) cycle. Reduced glucose metabolism correlated with a marked decrease in GLUT2 expression and several metabolic enzymes in unique brain regions. In conclusion, our study supports the incorporation of microwave fixation for more accurate studies of brain metabolism in rodent models.

Spatial metabolomics reveals glycogen as an actionable target for pulmonary fibrosis.

Matrix assisted laser desorption/ionization imaging has greatly improved our understanding of spatial biology, however a robust bioinformatic pipeline for data analysis is lacking. Here, we demonstrate the application of high-dimensionality reduction/spatial clustering and histopathological annotation of matrix assisted laser desorption/ionization imaging datasets to assess tissue metabolic heterogeneity in human lung diseases. Using metabolic features identified from this pipeline, we hypothesize that metabolic channeling between glycogen and N-linked glycans is a critical metabolic process favoring pulmonary fibrosis progression. To test our hypothesis, we induced pulmonary fibrosis in two different mouse models with lysosomal glycogen utilization deficiency. Both mouse models displayed blunted N-linked glycan levels and nearly 90% reduction in endpoint fibrosis when compared to WT animals. Collectively, we provide conclusive evidence that lysosomal utilization of glycogen is required for pulmonary fibrosis progression. In summary, our study provides a roadmap to leverage spatial metabolomics to understand foundational biology in pulmonary diseases.

Cervical spinal cord injury leads to injury and altered metabolism in the lungs.

High-cervical spinal cord injury often disrupts respiratory motor pathways and disables breathing in the affected population. Moreover, cervically injured individuals are at risk for developing acute lung injury, which predicts substantial mortality rates. While the correlation between acute lung injury and spinal cord injury has been found in the clinical setting, the field lacks an animal model to interrogate the fundamental biology of this relationship. To begin to address this gap in knowledge, we performed an experimental cervical spinal cord injury (N = 18) alongside sham injury (N = 3) and naïve animals (N = 15) to assess lung injury in adult rats. We demonstrate that animals display some early signs of lung injury two weeks post-spinal cord injury. While no obvious histological signs of injury were observed, the spinal cord injured cohort displayed significant signs of metabolic dysregulation in multiple pathways that include amino acid metabolism, lipid metabolism, and N-linked glycosylation. Collectively, we establish for the first time a model of lung injury after spinal cord injury at an acute time point that can be used to monitor the progression of lung damage, as well as identify potential targets to ameliorate acute lung injury.

PASK links cellular energy metabolism with a mitotic self-renewal network to establish differentiation competence.

Quiescent stem cells are activated in response to a mechanical or chemical injury to their tissue niche. Activated cells rapidly generate a heterogeneous progenitor population that regenerates the damaged tissues. While the transcriptional cadence that generates heterogeneity is known, the metabolic pathways influencing the transcriptional machinery to establish a heterogeneous progenitor population remains unclear. Here, we describe a novel pathway downstream of mitochondrial glutamine metabolism that confers stem cell heterogeneity and establishes differentiation competence by countering post-mitotic self-renewal machinery. We discovered that mitochondrial glutamine metabolism induces CBP/EP300-dependent acetylation of stem cell-specific kinase, PAS domain-containing kinase (PASK), resulting in its release from cytoplasmic granules and subsequent nuclear migration. In the nucleus, PASK catalytically outcompetes mitotic WDR5-anaphase-promoting complex/cyclosome (APC/C) interaction resulting in the loss of post-mitotic Pax7 expression and exit from self-renewal. In concordance with these findings, genetic or pharmacological inhibition of PASK or glutamine metabolism upregulated Pax7 expression, reduced stem cell heterogeneity, and blocked myogenesis in vitro and muscle regeneration in mice. These results explain a mechanism whereby stem cells co-opt the proliferative functions of glutamine metabolism to generate transcriptional heterogeneity and establish differentiation competence by countering the mitotic self-renewal network via nuclear PASK.

Metabolic modulation of synaptic failure and thalamocortical hypersynchronization with preserved consciousness in Glut1 deficiency.

Individuals with glucose transporter type I deficiency (G1D) habitually experience nutrient-responsive epilepsy associated with decreased brain glucose. However, the mechanistic association between blood glucose concentration and brain excitability in the context of G1D remains to be elucidated. Electroencephalography (EEG) in G1D individuals revealed nutrition time-dependent seizure oscillations often associated with preserved volition despite electrographic generalization and uniform average oscillation duration and periodicity, suggesting increased facilitation of an underlying neural loop circuit. Nonlinear EEG ictal source localization analysis and simultaneous EEG/functional magnetic resonance imaging converged on the thalamus-sensorimotor cortex as one potential circuit, and 18F-deoxyglucose positron emission tomography (18F-DG-PET) illustrated decreased glucose accumulation in this circuit. This pattern, reflected in a decreased thalamic to striatal 18F signal ratio, can aid with the PET imaging diagnosis of the disorder, whereas the absence of noticeable ictal behavioral changes challenges the postulated requirement for normal thalamocortical activity during consciousness. In G1D mice, 18F-DG-PET and mass spectrometry also revealed decreased brain glucose and glycogen, but preserved tricarboxylic acid cycle intermediates, indicating no overall energy metabolism failure. In brain slices from these animals, synaptic inhibition of cortical pyramidal neurons and thalamic relay neurons was decreased, and neuronal disinhibition was mitigated by metabolic sources of carbon; tonic-clonic seizures were also suppressed by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor inhibition. These results pose G1D as a thalamocortical synaptic disinhibition disease associated with increased glucose-dependent neuronal excitability, possibly in relation to reduced glycogen. Together with findings in other metabolic defects, inhibitory neuron dysfunction is emerging as a modulable mechanism of hyperexcitability.

In situ mass spectrometry imaging reveals heterogeneous glycogen stores in human normal and cancerous tissues.

Glycogen dysregulation is a hallmark of aging, and aberrant glycogen drives metabolic reprogramming and pathogenesis in multiple diseases. However, glycogen heterogeneity in healthy and diseased tissues remains largely unknown. Herein, we describe a method to define spatial glycogen architecture in mouse and human tissues using matrix-assisted laser desorption/ionization mass spectrometry imaging. This assay provides robust and sensitive spatial glycogen quantification and architecture characterization in the brain, liver, kidney, testis, lung, bladder, and even the bone. Armed with this tool, we interrogated glycogen spatial distribution and architecture in different types of human cancers. We demonstrate that glycogen stores and architecture are heterogeneous among diseases. Additionally, we observe unique hyperphosphorylated glycogen accumulation in Ewing sarcoma, a pediatric bone cancer. Using preclinical models, we correct glycogen hyperphosphorylation in Ewing sarcoma through genetic and pharmacological interventions that ablate in vivo tumor growth, demonstrating the clinical therapeutic potential of targeting glycogen in Ewing sarcoma.

Tissue-Specific Downregulation of Fatty Acid Synthase Suppresses Intestinal Adenoma Formation via Coordinated Reprograming of Transcriptome and Metabolism in the Mouse Model of Apc-Driven Colorectal Cancer.

Altered lipid metabolism is a potential target for therapeutic intervention in cancer. Overexpression of Fatty Acid Synthase (FASN) correlates with poor prognosis in colorectal cancer (CRC). While multiple studies show that upregulation of lipogenesis is critically important for CRC progression, the contribution of FASN to CRC initiation is poorly understood. We utilize a C57BL/6-Apc/Villin-Cre mouse model with knockout of FASN in intestinal epithelial cells to show that the heterozygous deletion of FASN increases mouse survival and decreases the number of intestinal adenomas. Using RNA-Seq and gene set enrichment analysis, we demonstrate that a decrease in FASN expression is associated with inhibition of pathways involved in cellular proliferation, energy production, and CRC progression. Metabolic and reverse phase protein array analyses demonstrate consistent changes in alteration of metabolic pathways involved in both anabolism and energy production. Downregulation of FASN expression reduces the levels of metabolites within glycolysis and tricarboxylic acid cycle with the most significant reduction in the level of citrate, a master metabolite, which enhances ATP production and fuels anabolic pathways. In summary, we demonstrate the critical importance of FASN during CRC initiation. These findings suggest that targeting FASN is a potential therapeutic approach for early stages of CRC or as a preventive strategy for this disease.

The Toxoplasma glucan phosphatase TgLaforin utilizes a distinct functional mechanism that can be exploited by therapeutic inhibitors.

Toxoplasma gondii is an intracellular parasite that generates amylopectin granules (AGs), a polysaccharide associated with bradyzoites that define chronic T. gondii infection. AGs are postulated to act as an essential energy storage molecule that enable bradyzoite persistence, transmission, and reactivation. Importantly, reactivation can result in the life-threatening symptoms of toxoplasmosis. T. gondii encodes glucan dikinase and glucan phosphatase enzymes that are homologous to the plant and animal enzymes involved in reversible glucan phosphorylation and which are required for efficient polysaccharide degradation and utilization. However, the structural determinants that regulate reversible glucan phosphorylation in T. gondii are unclear. Herein, we define key functional aspects of the T. gondii glucan phosphatase TgLaforin (TGME49_205290). We demonstrate that TgLaforin possesses an atypical split carbohydrate-binding-module domain. AlphaFold2 modeling combined with hydrogen-deuterium exchange mass spectrometry and differential scanning fluorimetry also demonstrate the unique structural dynamics of TgLaforin with regard to glucan binding. Moreover, we show that TgLaforin forms a dual specificity phosphatase domain-mediated dimer. Finally, the distinct properties of the glucan phosphatase catalytic domain were exploited to identify a small molecule inhibitor of TgLaforin catalytic activity. Together, these studies define a distinct mechanism of TgLaforin activity, opening up a new avenue of T. gondii bradyzoite biology as a therapeutic target.