Inorganic phosphate (Pi) is an essential nutrient for the human body which exerts adverse health effects in excess and deficit. High Pi-mediated cytotoxicity has been shown to induce systemic organ damage, though the underlying molecular mechanisms are poorly understood. In this study, we employed proteomics and phosphoproteomics to analyze Pi-mediated changes in protein abundance and phosphorylation. Bioinformatic analyses and literature review revealed that the altered proteins and phosphorylation were enriched in signaling pathways and diverse biological processes. Western blot analysis confirms the extensive change in protein level and phosphorylation in key effectors that modulate pre-mRNA alternative splicing. Global proteome and phospho-profiling provide a bird-eye view of excessive Pi-rewired cell signaling networks, which deepens our understanding of the molecular mechanisms of phosphate toxicity.

The underlying role of inadequate or excess intake of phosphate is evident in disease states, including metabolic, skeletal, cardiac, kidney and various cancers. Elevated phosphate levels can induce epithelial to mesenchymal transition (EMT) and cell death. EMT and associated lethal, metastatic or fibrinogenic responses are known to be underlying disease processes in fibrotic diseases and various solid tumors. Studies have shown EMT is regulated by induction of different signaling pathways, including TGF-β, RTK, SRC, Wnt and Notch signal transduction. However, cross-talk amongst these signaling pathways is less understood. We have shown that elevated phosphate levels enhanced EMT partially through activating ERK1/2 pathway, resulting in massive cell death. We thus proposed excess phosphate-mediated lethal EMT as one of the underlying mechanisms of phosphate-induced cytotoxicity, which could explain high phosphate-associated renal fibrosis and cancer metastasis in preclinical and clinical studies. This chapter provides the overview of EMT with the highlights of its regulation by various signaling pathways induced by phosphate toxicity. We further put lately reported lethal EMT in the context of phosphate toxicity with the intent to explain it to excessive phosphate-associated pathologies.

N-glycanase 1(NGLY1) catalyzes the removal of N-linked glycans from newly synthesized or misfolded protein. NGLY1 deficiency is a recently diagnosed rare genetic disorder. The affected individuals present a broad spectrum of clinical features. Recent studies explored several possible molecular mechanisms of NGLY1 deficiency including defects in proteostasis, mitochondrial homeostasis, innate immunity, and water/ion transport. We demonstrate abnormal accumulation of endoplasmic reticulum-associated degradation (ERAD) substrates in NGLY1-deficient cells. Global quantitative proteomics discovered elevated levels of endogenous proteins in NGLY1-defective human and mouse cells. Further biological validation assays confirmed the altered abundance of several key candidates that were subjected to isobarically labeled proteomic analysis. CCN2 was selected for further analysis due to its significant increase in different cell models of NGLY1 deficiency. Functional assays show elevated CCN2 and over-stimulated TGF-β signaling in NGLY1-deficient cells. Given the important role of CCN2 and TGF-β pathway in mediating systemic fibrosis, we propose a potential link of increased CCN2 and TGF-β signaling to microscopic liver fibrosis in NGLY1 patients.

Phosphorus (usually as phosphate) is an essential nutrient used for enzyme activation and the synthesis of adenosine triphosphate and other biomolecules. Most phosphorus in the body is sequestered in mineralized bone tissue and teeth. Phosphate homeostasis is maintained by FGF23 derived from bones and the kidney-derived cofactor Klotho. FGF23 binds the membrane-bound Klotho–FGF receptor complex with much higher affinity than the FGF receptor (FGFR) alone to generate downstream signaling events. Aberrations in FGF23, FGFR, or Klotho structure or expression result in dysregulation of serum phosphate levels in experimental animal models and human diseases. The FGF23–Klotho system suppresses sodium-phosphate cotransporters (NaPi2 family proteins) in proximal tubular epithelial cells in the kidney to induce urinary phosphate wasting. In addition, the FGF23–Klotho system also regulates vitamin D metabolism, which is an important factor that regulates the homeostasis of minerals such as calcium, phosphorus, and magnesium. FGF23 can reduce the activity of 1,25-dihydroxyvitamin D3 by increasing the synthesis of the catabolic enzyme 24-hydroxylase and by suppressing renal 1-α hydroxylase expression. Determination of FGF23 or Klotho status may aid in the diagnosis and prognosis of diseases associated with abnormal phosphate levels such as chronic kidney disease, cardiac dysfunction, and bone mineralization defects. Preclinical and clinical studies suggest that dietary phosphate overload has toxic and prolonged adverse health effects. There are pharmaceuticals and biomedical devices to mitigate absorption (or reabsorption) of phosphate from food (or blood) into the body, but these strategies have limited efficacy. Without measures in place to reduce dietary phosphate intake, the conditions associated with phosphate toxicity will likely become a global health concern.

Phosphate, an essential nutrient, is available in organic and inorganic forms. The balance of phosphate is central for cellular homeostasis through the genomic roles of DNA and RNA synthesis and cell signaling processes. Therefore, an imbalance of this nutrient, manifested, either as a deficiency or excess in phosphate levels, can result in pathology, ranging from cytotoxicity to musculoskeletal defects. Inorganic phosphate (Pi) overdosing can result in a wide spectrum of cytotoxicity processes, as noted in both animal models and human studies. These include rewired cell signaling pathways, impaired bone mineralization, infertility, premature aging, vascular calcification, and renal dysfunction. This article briefly reviews the regulation of phosphate homeostasis and elaborates on cytotoxic effects of excessive Pi, as documented in cell-based models.

Inorganic phosphate (Pi) is an essential nutrient for human health. Due to the changes in our dietary pattern, dietary Pi overload engenders systemic phosphotoxicity, including excessive Pi-related vascular calcification and chronic tissue injury. The molecular mechanisms of the seemingly distinct phenotypes remain elusive. In this study, we investigated Pi-mediated cellular response in HEK293 and HeLa cells. We found that abnormally high Pi directly mediates diverse cellular toxicity in a dose-dependent manner. Up to 10 mM extracellular Pi promotes cell proliferation by activating AKT signaling cascades and augmenting cell cycle progression. By introducing additional Pi, higher than the concentration of 40 mM, we observed significant cell damage caused by the interwoven Pi-related biological processes. Elevated Pi activates mitogen-activated protein kinase (MAPK) signaling, encompassing extracellular signal-regulated kinase 1/2 (ERK1/2), p38 and Jun amino-terminal kinase (JNK), which consequently potentiates Pi triggered lethal epithelial-mesenchymal transition (EMT). Synergistically, high Pi-caused endoplasmic reticulum (ER) stress also contributes to apparent apoptosis. To counteract, Pi-activated AKT signaling promotes cell survival by activating the mammalian target of rapamycin (mTOR) signaling and blocking ER stress. Pharmacologically or genetically abrogating Pi transport, the impact of high Pi-induced cytotoxicity could be reduced. Taken together, abnormally high extracellular Pi results in a broad spectrum of toxicity by rewiring complicated signaling networks that control cell growth, cell death, and homeostasis.

OBJECTIVE:Evidence suggested that traumatic events, including pandemics, can be associated with psychiatric symptoms like increased anxiety and depression. However, there were many unknowns concerning the emergent global coronavirus-19 (COVID-19), including its impact on psychiatric health within the United States. Our study aimed to track trends of mental health problems in individuals who presented with psychiatric complaints in an emergent setting. METHODS:A total of 1776 patients and 1610 patients presented to Emergency Department (ED) with psychiatric complaints between January 1 - July 9 of the years of 2019 and 2020, respectively, in Millcreek Community Hospital (MCH) Erie, PA. This study was an electronic medical record review (EMR), therefore the data were collected exclusively from EMR over the two-year span. ED prevalence was calculated as the number of total psychiatric MCH ED cases divided by the total number of all MCH ED patients, and prevalence ratio (PR) between 2019 and 2020 was used to reflect change of overall ED psychiatric prevalence. RESULTS:Clinical notes revealed increased ED psychiatric chief complaint prevalence, as indicated by a PR greater than one, in multiple categories in comparison to before the COVID-19 outbreak. Concerning primary psychiatric disorders, there was increased ED prevalence in chief complaint of total mood disorders (PR = 1.21) with major depressive disorder (PR = 1.23) and bipolar disorder (PR = 1.47), neurodevelopment disorders (PR = 1.25) with attention deficit hyperactivity disorder (ADHD) (PR = 1.19) and intellectual disability (PR = 1.52), trauma- and stressor-related disorders (PR = 1.56) with post-traumatic stress disorder (PTSD) (PR = 1.39) and adjustment disorder (PR = 1.73), substance abuse and addiction disorders (PR = 1.29), and personality disorders (PR = 1.56). CONCLUSIONS:The pandemic outbreak dramatically impacted mental health in an ER setting. Further research on mental health disparities in conjunction with the COVID-19 pandemic is critical to help predict and address risk for chronic symptoms and sequela to help anticipate and improve psychiatric patient care and well-being during potential future pandemics.

Inorganic phosphate (Pi) is an essential nutrient for human health. Due to our change in dietary pattern, dietary Pi overload engenders systematic phosphotoxicity, including excessive Pi related vascular calcification and chronic tissue injury. The molecular mechanisms of the seemingly distinct phenotypes remain elusive. In this study, we found that Pi directly mediates diverse cellular toxicity in a dose-dependent manner on a cell-based model. At moderately higher than physiological level, extracellular Pi promotes cell proliferation by activating AKT and extracellular signal-regulated kinase 1/2 (ERK1/2) cascades. By introducing additional Pi, we observed significant cell damage caused by the interwoven Pi related biological processes, including activation of mitogen-activated protein kinase (MAPK) signaling, endoplasmic reticulum (ER) stress, epithelial-mesenchymal transition (EMT) and apoptosis. Taken together, elevated extracellular Pi results in a broad spectrum of toxicity by rewiring complicated signaling networks that control cell growth, cell death, ER stress, and cell mobility.

N-Glycanase 1, encoded by NGLY1, catalyzes the deglycosylation of misfolded N-linked glycoproteins retrotranslocated into the cytosol.We identified nine cases with mutations in NGLY1.The patients show developmental delay, seizures, peripheral neuropathy, abnormal liver function and alacrima (absence of tears).The mutations in NGLY1 resulted in the absence of N-glycanase 1 protein in patient-derived fibroblasts.Applying a recently established cellular deglycosylationdependent Venus fluorescence assay, we found that patient fibroblasts had dramatically reduced fluorescence, indicating a pronounced reduction in N-glycanase enzymatic activity.Using this assay, we could find no evidence of other related activities.Our findings reveal that NGLY1 mutations destroy both N-glycanase 1 protein and enzymatic activity.

Patients with congenital disorder of glycosylation (CDG), type Ib (MPI‐CDG or CDG‐Ib) have mutations in phosphomannose isomerase (MPI) that impair glycosylation and lead to stunted growth, liver dysfunction, coagulopathy, hypoglycemia, and intestinal abnormalities. Mannose supplements correct hypo‐glycosylation and most symptoms by providing man‐nose‐6‐P (Man‐6‐P) via hexokinase. We generated viable Mpi hypomorphic mice with residual enzymatic activity comparable to that of patients, but surprisingly, these mice appeared completely normal except for modest (~15%) embryonic lethality. To overcome this lethality, pregnant dams were provided 1–2% mannose in their drinking water. However, mannose further reduced litter size and survival to weaning by 40 and 66%, respectively. Moreover, ~50% of survivors developed eye defects beginning around midgestation. Mannose started at birth also led to eye defects but had no effect when started after eye development was complete. Man‐6‐P and related metabolites accumulated in the affected adult eye and in developing embryos and placentas. Our results demonstrate that disturbing mannose metabolic flux in mice, especially during embryonic development, induces a highly specific, unanticipated pathological state. It is unknown whether mannose is harmful to human fetuses during gestation; however, mothers who are at risk for having MPI‐CDG children and who consume mannose during pregnancy hoping to benefit an affected fetus in utero should be cautious.—Sharma, V., Nayak, J., DeRossi, C., Charbono, A., Ichikawa, M., Ng, B. G., Grajales‐Esquivel, E., Srivastava, A., Wang, L., He, P., Scott, D. A., Russell, J., Contreras, E., Guess, C. M., Krajewski, S., Del Rio‐Tsonis, K., Freeze, H. H. Mannose supplements induce embryonic lethality and blindness in phosphomannose isomerase hypomorphic mice. FASEB J. 28, 1854–1869 (2014). www.fasebj.org

PURPOSE:The endoplasmic reticulum-associated degradation pathway is responsible for the translocation of misfolded proteins across the endoplasmic reticulum membrane into the cytosol for subsequent degradation by the proteasome. To define the phenotype associated with a novel inherited disorder of cytosolic endoplasmic reticulum-associated degradation pathway dysfunction, we studied a series of eight patients with deficiency of N-glycanase 1.METHODS:Whole-genome, whole-exome, or standard Sanger sequencing techniques were employed. Retrospective chart reviews were performed in order to obtain clinical data.RESULTS:All patients had global developmental delay, a movement disorder, and hypotonia. Other common findings included hypolacrima or alacrima (7/8), elevated liver transaminases (6/7), microcephaly (6/8), diminished reflexes (6/8), hepatocyte cytoplasmic storage material or vacuolization (5/6), and seizures (4/8). The nonsense mutation c.1201A>T (p.R401X) was the most common deleterious allele.CONCLUSION:NGLY1 deficiency is a novel autosomal recessive disorder of the endoplasmic reticulum-associated degradation pathway associated with neurological dysfunction, abnormal tear production, and liver disease. The majority of patients detected to date carry a specific nonsense mutation that appears to be associated with severe disease. The phenotypic spectrum is likely to enlarge as cases with a broader range of mutations are detected.

Congenital Disorders of Glycosylation (CDGs) result from mutations in various N‐glycosylation genes. Many patients are prone to infections, but the mechanism is unknown. We found that CDG patient fibroblasts have less intercellular adhesion molecule‐1 (ICAM‐1), and because of its role in innate immune response, we hypothesized that its reduction may explain recurrent infections. In support of this, tunicamycin blocks TNF‐α induced ICAM‐1 production, suggesting that many CDG cells may show poor ICAM‐1 response. We, therefore, investigated a mouse model of MPI‐CDG, deficient in phosphomannose isomerase. We challenged MPI‐deficient mice with an intraperitoneal injection of pro‐inflammatory mediator (Zymosan) and found decreased neutrophil extravasation. Immunohistochemical staining of mesenteries showed attenuated neutrophil egress, presumably due to poor ICAM‐1 response to acute peritonitis. Since MPI‐CDG patients and their cells improve glycosylation when given mannose, we provided MPI‐deficient mice with mannose‐supplemented water for 7 days. This increased ICAM‐1 expression on mesenteric endothelial cells of Zymosan‐challenged mice and enhanced neutrophils exudation compared to untreated controls. Attenuated inflammatory response in glycosylation‐deficient mice may result from ICAM‐1 deficiency on the vascular endothelial surface and may help explain high mortality in patients.Grant Funding Source: supported by the Rocket Fund & NIH R01 DK55615

Congenital disorders of glycosylation (CDGs) result from mutations in various N-glycosylation genes. The most common type, phosphomannomutase-2 (PMM2)-CDG (CDG-Ia), is due to deficient PMM2 (Man-6-P → Man-1-P). Many patients die from recurrent infections, but the mechanism is unknown. We found that glycosylation-deficient patient fibroblasts have less intercellular adhesion molecule-1 (ICAM-1), and because of its role in innate immune response, we hypothesized that its reduction might help explain recurrent infections in CDG patients. We, therefore, studied mice with mutations in Mpi encoding phosphomannose isomerase (Fru-6-P → Man-6-P), the cause of human MPI-CDG. We challenged MPI-deficient mice with an intraperitoneal injection of zymosan to induce an inflammatory response and found decreased neutrophil extravasation compared with control mice. Immunohistochemistry of mesenteries showed attenuated neutrophil egress, presumably due to poor ICAM-1 response to acute peritonitis. Since phosphomannose isomerase (MPI)-CDG patients and their cells improve glycosylation when given mannose, we provided MPI-deficient mice with mannose-supplemented water for 7 days. This restored ICAM-1 expression on mesenteric endothelial cells and enhanced transendothelial migration of neutrophils during acute inflammation. Attenuated inflammatory response in glycosylation-deficient mice may result from a failure to increase ICAM-1 on the vascular endothelial surface and may help explain recurrent infections in patients.

N‐glycanase 1, encoded by NGLY1, catalyzes the deglycosylation of misfolded N‐linked glycoproteins. Using whole‐genome and ‐exome sequencing, we identified 6 cases with mutations in NGLY1. The patients show developmental delay, seizures, peripheral neuropathy, abnormal liver function, and absent tears. The mutations in NGLY1 resulted in drastic reduction of N‐glycanase 1 protein in patient‐derived fibroblasts. We probed N‐glycanase 1 enzymatic activity firstly by measuring the enzyme released free oligosaccharides (fOS). The patient fibroblasts produced 2‐3 folds less fOS and showed an altered size distribution. Applying a recently established cellular deglycosylation dependent Venus fluorescence assay, we found that patient fibroblasts had dramatically reduced fluorescence indicating pronounced enzymatic activity reduction in N‐glycanase 1. Since deglycosylation of misfolded glycoproteins precedes proteasomal degradation, we speculated that N‐glycanase 1 deficiency might also accumulate misfolded glycoproteins, leading to ER stress. We observed 5‐10 fold increase of transfected ERAD substrates accumulation in the cytoplasm. However, under physiological conditions, we did not observe abnormalities of the three branches of UPR signal transduction (IRE1, PERK, and ATF6) in patient fibroblasts, indicating that the N‐glycanase 1 deficiency itself is not sufficient to cause ER stress.Grant Funding Source: Supported by the Bertrand Might Research Fund

Time 8:30 Glycobiology of Human Pluripotent Stem Cells; Steve Dalton, University of Georgia #1 9:00 Siglec-Sialoglycan Binding Regulates Cell-Cell Interactions; Ronald Schnaar, Johns Hopkins School of Medicine 9:30 Stem Cell Therapies in Epidermolysis Bullosa; Angela Christiano, Columbia University Medical Center 10:00 – 10:30 am Coffee Break (Grande Foyer and Grande Ballroom A) 10:30 am – 12:00 pm Plenary II: Biomaterials and Matrix Engineering (Grande Ballroom B & C) Chair: Adam Engler, University of California, San Diego 10:30 Hydrogels as Synthetic Extracellular Matrices; Kristi Anseth, University of Colorado at Boulder 11:00 Mechanical Regulation of Cell Adhesion and Function; Christopher Chen, University of Pennsylvania Joint Meeting of the Society for Glycobiology & American Society for Matrix Biology Conference Program by A rm en Peosyan on N ovem er 2, 2012 http://glycfordjournals.org/ D ow nladed from