Metabolic Bypass Rescues Aberrant S-nitrosylation-Induced TCA Cycle Inhibition and Synapse Loss in Alzheimer's Disease Human Neurons.

In Alzheimer's disease (AD), dysfunctional mitochondrial metabolism is associated with synaptic loss, the major pathological correlate of cognitive decline. Mechanistic insight for this relationship, however, is still lacking. Here, comparing isogenic wild-type and AD mutant human induced pluripotent stem cell (hiPSC)-derived cerebrocortical neurons (hiN), evidence is found for compromised mitochondrial energy in AD using the Seahorse platform to analyze glycolysis and oxidative phosphorylation (OXPHOS). Isotope-labeled metabolic flux experiments revealed a major block in activity in the tricarboxylic acid (TCA) cycle at the α-ketoglutarate dehydrogenase (αKGDH)/succinyl coenzyme-A synthetase step, metabolizing α-ketoglutarate to succinate. Associated with this block, aberrant protein S-nitrosylation of αKGDH subunits inhibited their enzyme function. This aberrant S-nitrosylation is documented not only in AD-hiN but also in postmortem human AD brains versus controls, as assessed by two separate unbiased mass spectrometry platforms using both SNOTRAP identification of S-nitrosothiols and chemoselective-enrichment of S-nitrosoproteins. Treatment with dimethyl succinate, a cell-permeable derivative of a TCA substrate downstream to the block, resulted in partial rescue of mitochondrial bioenergetic function as well as reversal of synapse loss in AD-hiN. These findings have therapeutic implications that rescue of mitochondrial energy metabolism can ameliorate synaptic loss in hiPSC-based models of AD.

Stratification of neurogenic bladder risk in spina bifida using the urinary peptidome.

Neurogenic bladder poses a major morbidity in children with spina bifida (SB), and videourodynamic studies (VUDS) are used to stratify this risk. This small-scale pilot study utilized current mass-spectrometry-based proteomic approaches to identify peptides or proteins in urine that may differentiate children at high risk of developing renal complications from a neurogenic bladder. Twenty-two urine samples of which nine had high bladder pressure storage that put the upper urinary tract at risk, while 13 with a lower risk for renal compromise were analyzed. More than 1,900 peptides across all 22 samples were quantified, and 115 peptides differed significantly (P < 0.05) between the two groups. Using machine learning approaches five peptides that showed the greatest differences between these two clinical categories were used to build a classifier. We tested this classifier by blind analysis of an additional six urine samples and showed that it correctly assigned the unknown samples in their proper risk category. These promising results indicate that a urinary screening test based on peptides could be performed on a regular basis to stratify the neurogenic bladder into low or high-risk categories. Expanding this work to larger cohorts as well as across a broad spectrum of urodynamics outcomes may provide a useful diagnostic test for neurogenic bladder.NEW & NOTEWORTHY This approach could help risk stratify the neurogenic bladder in patients with spina bifida and could allow us to safely defer on up to 1/3 of urodynamic studies. These pilot data justify a larger trial before this approach becomes a clinical tool.

Quantitative label-free mass spectrometry reveals content and signaling differences between neonatal and adult platelets.

BACKGROUND: Recent clinical studies have shown that transfusions of adult platelets increase morbidity and mortality in preterm infants. Neonatal platelets are hyporesponsive to agonist stimulation, and emerging evidence suggests developmental differences in platelet immune functions. OBJECTIVES: This study was designed to compare the proteome and phosphoproteome of resting adult and neonatal platelets. METHODS: We isolated resting umbilical cord blood-derived platelets from healthy full-term neonates (n = 8) and resting blood platelets from healthy adults (n = 6) and compared protein and phosphoprotein contents using data-independent acquisition mass spectrometry. RESULTS: We identified 4770 platelet proteins with high confidence across all samples. Adult and neonatal platelets were clustered separately by principal component analysis. Adult platelets were significantly enriched in immunomodulatory proteins, including ß2 microglobulin and CXCL12, whereas neonatal platelets were enriched in ribosomal components and proteins involved in metabolic activities. Adult platelets were enriched in phosphorylated GTPase regulatory enzymes and proteins participating in trafficking, which may help prime them for activation and degranulation. Neonatal platelets were enriched in phosphorylated proteins involved in insulin growth factor signaling. CONCLUSION: Using label-free data-independent acquisition mass spectrometry, our findings expanded the known neonatal platelet proteome and identified important differences in protein content and phosphorylation between neonatal and adult platelets. These developmental differences suggested enhanced immune functions for adult platelets and presence of molecular machinery related to platelet activation. These findings are important to understanding mechanisms underlying key platelet functions as well as the harmful effects of adult platelet transfusions given to preterm infants.

Phosphoproteomics reveals content and signaling differences between neonatal and adult platelets.

Background and Objective: Recent clinical studies have shown that transfusions of adult platelets increase morbidity and mortality in preterm infants. Neonatal platelets are hyporesponsive to agonist stimulation, and emerging evidence suggests developmental differences in platelet immune functions. This study was designed to compare the proteome and phosphoproteome of resting adult and neonatal platelets. Methods: We isolated resting umbilical cord blood-derived platelets from healthy full term neonates (n=9) and resting blood platelets from healthy adults (n=7), and compared protein and phosphoprotein contents using data independent acquisition mass spectrometry. Results: We identified 4745 platelet proteins with high confidence across all samples. Adult and neonatal platelets clustered separately by principal component analysis. Adult platelets were significantly enriched for immunomodulatory proteins, including ß2 microglobulin and CXCL12, whereas neonatal platelets were enriched for ribosomal components and proteins involved in metabolic activities. Adult platelets were enriched for phosphorylated GTPase regulatory enzymes and proteins participating in trafficking, which may help prime them for activation and degranulation. Neonatal platelets were enriched for phosphorylated proteins involved in insulin growth factor signaling. Conclusions: Using state-of-the-art mass spectrometry, our findings expanded the known neonatal platelet proteome and identified important differences in protein content and phosphorylation compared with adult platelets. These developmental differences suggested enhanced immune functions for adult platelets and presence of a molecular machinery related to platelet activation. These findings are important to understanding mechanisms underlying key platelet functions as well as the harmful effects of adult platelet transfusions given to preterm infants.

S-Nitrosylation-mediated dysfunction of TCA cycle enzymes in synucleinopathy studied in postmortem human brains and hiPSC-derived neurons.

A causal relationship between mitochondrial metabolic dysfunction and neurodegeneration has been implicated in synucleinopathies, including Parkinson disease (PD) and Lewy body dementia (LBD), but underlying mechanisms are not fully understood. Here, using human induced pluripotent stem cell (hiPSC)-derived neurons with mutation in the gene encoding α-synuclein (αSyn), we report the presence of aberrantly S-nitrosylated proteins, including tricarboxylic acid (TCA) cycle enzymes, resulting in activity inhibition assessed by carbon-labeled metabolic flux experiments. This inhibition principally affects α-ketoglutarate dehydrogenase/succinyl coenzyme-A synthetase, metabolizing α-ketoglutarate to succinate. Notably, human LBD brain manifests a similar pattern of aberrantly S-nitrosylated TCA enzymes, indicating the pathophysiological relevance of these results. Inhibition of mitochondrial energy metabolism in neurons is known to compromise dendritic length and synaptic integrity, eventually leading to neuronal cell death. Our evidence indicates that aberrant S-nitrosylation of TCA cycle enzymes contributes to this bioenergetic failure.

Unveiling the human nitroproteome: Protein tyrosine nitration in cell signaling and cancer.

Covalent amino acid modification significantly expands protein functional capability in regulating biological processes. Tyrosine residues can undergo phosphorylation, sulfation, adenylation, halogenation, and nitration. These posttranslational modifications (PTMs) result from the actions of specific enzymes: tyrosine kinases, tyrosyl-protein sulfotransferase(s), adenylate transferase(s), oxidoreductases, peroxidases, and metal-heme containing proteins. Whereas phosphorylation, sulfation, and adenylation modify the hydroxyl group of tyrosine, tyrosine halogenation and nitration target the adjacent carbon residues. Because aberrant tyrosine nitration has been associated with human disorders and with animal models of disease, we have created an updated and curated database of 908 human nitrated proteins. We have also analyzed this new resource to provide insight into the role of tyrosine nitration in cancer biology, an area that has not previously been considered in detail. Unexpectedly, we have found that 879 of the 1971 known sites of tyrosine nitration are also sites of phosphorylation suggesting an extensive role for nitration in cell signaling. Overall, the review offers several forward-looking opportunities for future research and new perspectives for understanding the role of tyrosine nitration in cancer biology.

Nitrative Modification of Caveolin-3: A Novel Mechanism of Cardiac Insulin Resistance and a Potential Therapeutic Target Against Ischemic Heart Failure in Prediabetic Animals.

BACKGROUND: Myocardial insulin resistance is a hallmark of diabetic cardiac injury. However, the underlying molecular mechanisms remain unclear. Recent studies demonstrate that the diabetic heart is resistant to other cardioprotective interventions, including adiponectin and preconditioning. The "universal" resistance to multiple therapeutic interventions suggests impairment of the requisite molecule(s) involved in broad prosurvival signaling cascades. Cav (Caveolin) is a scaffolding protein coordinating transmembrane signaling transduction. However, the role of Cav3 in diabetic impairment of cardiac protective signaling and diabetic ischemic heart failure is unknown. METHODS: Wild-type and gene-manipulated mice were fed a normal diet or high-fat diet for 2 to 12 weeks and subjected to myocardial ischemia and reperfusion. Insulin cardioprotection was determined. RESULTS: Compared with the normal diet group, the cardioprotective effect of insulin was significantly blunted as early as 4 weeks of high-fat diet feeding (prediabetes), a time point where expression levels of insulin-signaling molecules remained unchanged. However, Cav3/insulin receptor-ß complex formation was significantly reduced. Among multiple posttranslational modifications altering protein/protein interaction, Cav3 (not insulin receptor-ß) tyrosine nitration is prominent in the prediabetic heart. Treatment of cardiomyocytes with 5-amino-3-(4-morpholinyl)-1,2,3-oxadiazolium chloride reduced the signalsome complex and blocked insulin transmembrane signaling. Mass spectrometry identified Tyr73 as the Cav3 nitration site. Phenylalanine substitution of Tyr73 (Cav3Y73F) abolished 5-amino-3-(4-morpholinyl)-1,2,3-oxadiazolium chloride-induced Cav3 nitration, restored Cav3/insulin receptor-ß complex, and rescued insulin transmembrane signaling. It is most important that adeno-associated virus 9-mediated cardiomyocyte-specific Cav3Y73F reexpression blocked high-fat diet-induced Cav3 nitration, preserved Cav3 signalsome integrity, restored transmembrane signaling, and rescued insulin-protective action against ischemic heart failure. Last, diabetic nitrative modification of Cav3 at Tyr73 also reduced Cav3/AdipoR1 complex formation and blocked adiponectin cardioprotective signaling. CONCLUSIONS: Nitration of Cav3 at Tyr73 and resultant signal complex dissociation results in cardiac insulin/adiponectin resistance in the prediabetic heart, contributing to ischemic heart failure progression. Early interventions preserving Cav3-centered signalsome integrity is an effective novel strategy against diabetic exacerbation of ischemic heart failure.

The effects of a motorized passive simulated jogging device on descent of the arterial pulse waveform dicrotic notch: A single arm placebo-controlled cross-over trial.

Whole Body Periodic Acceleration (WBPA, pGz), is a bed that moves the body headward to forward, adds pulses to the circulation inducing descent of the dicrotic notch (DN) on the pulse waveform with an increase in a/b ratio (a = the height of the pulse waveform and b = the height of the secondary wave). Since the WBPA is large, heavy, and non-portable, we engineered a portable device (Jogging Device, JD). JD simulates passive jogging and introduces pulsations to the circulation. We hypothesized that JD would increase the a/b ratio during and after its use. In Study A, a single-arm placebo-controlled cross-over trial was conducted in24 adults (53.8 ± 14.4 years) using JD or control (CONT) for 30 min. Blood pressure (BPs and BPd) and photoplethysmograph pulse (a/b) were measured at baseline (BL), during 30 min of JD or CONT, and 5 and 60 min after. In Study B (n = 20, 52.2 ± 7 years), a single-arm observational trial of 7 consecutive days of JD on BP and a/b, measured at BL, and after 7 days of JD and 48 and 72 hr after its discontinuation. In Study A, BPs, and BPd decreased during JD by 13% and 16%, respectively, while in CONT both increased by 2% and 2.5%, respectively. The a/b increased by 2-fold and remained greater than 2-fold at all-time points, with no change in a/b during CONT. In Study B, BPs and BPd decreased by 9% and remained below BL, at 72 hr after discontinuation of JD. DN descent also occurred after 7 days of JD with a/b increase of 80% and remained elevated by 60% for at least 72 h. JD improves acute and longer-term vascular hemodynamics with an increase in a/b, consistent with increased effects of nitric oxide (NO). JD may have significant clinical and public health implications.

PPARdelta activation induces metabolic and contractile maturation of human pluripotent stem cell-derived cardiomyocytes.

Pluripotent stem-cell-derived cardiomyocytes (PSC-CMs) provide an unprecedented opportunity to study human heart development and disease, but they are functionally and structurally immature. Here, we induce efficient human PSC-CM (hPSC-CM) maturation through metabolic-pathway modulations. Specifically, we find that peroxisome-proliferator-associated receptor (PPAR) signaling regulates glycolysis and fatty acid oxidation (FAO) in an isoform-specific manner. While PPARalpha (PPARa) is the most active isoform in hPSC-CMs, PPARdelta (PPARd) activation efficiently upregulates the gene regulatory networks underlying FAO, increases mitochondrial and peroxisome content, enhances mitochondrial cristae formation, and augments FAO flux. PPARd activation further increases binucleation, enhances myofibril organization, and improves contractility. Transient lactate exposure, which is frequently used for hPSC-CM purification, induces an independent cardiac maturation program but, when combined with PPARd activation, still enhances oxidative metabolism. In summary, we investigate multiple metabolic modifications in hPSC-CMs and identify a role for PPARd signaling in inducing the metabolic switch from glycolysis to FAO in hPSC-CMs.

ASS1 and ASL suppress growth in clear cell renal cell carcinoma via altered nitrogen metabolism.

BACKGROUND: Kidney cancer is a common adult malignancy in the USA. Clear cell renal cell carcinoma (ccRCC), the predominant subtype of kidney cancer, is characterized by widespread metabolic changes. Urea metabolism is one such altered pathway in ccRCC. The aim of this study was to elucidate the contributions of urea cycle enzymes, argininosuccinate synthase 1 (ASS1), and argininosuccinate lyase (ASL) towards ccRCC progression. METHODS: We employed a combination of computational, genetic, and metabolomic tools along with in vivo animal models to establish a tumor-suppressive role for ASS1 and ASL in ccRCC. RESULTS: We show that the mRNA and protein expression of urea cycle enzymes ASS1 and ASL are reduced in ccRCC tumors when compared to the normal kidney. Furthermore, the loss of ASL in HK-2 cells (immortalized renal epithelial cells) promotes growth in 2D and 3D growth assays, while combined re-expression of ASS1 and ASL in ccRCC cell lines suppresses growth in 2D, 3D, and in vivo xenograft models. We establish that this suppression is dependent on their enzymatic activity. Finally, we demonstrate that conservation of cellular aspartate, regulation of nitric oxide synthesis, and pyrimidine production play pivotal roles in ASS1+ASL-mediated growth suppression in ccRCC. CONCLUSIONS: ccRCC tumors downregulate the components of the urea cycle including the enzymes argininosuccinate synthase 1 (ASS1) and argininosuccinate lyase (ASL). These cytosolic enzymes lie at a critical metabolic hub in the cell and are involved in aspartate catabolism and arginine and nitric oxide biosynthesis. Loss of ASS1 and ASL helps cells redirect aspartate towards pyrimidine synthesis and support enhanced proliferation. Additionally, reduced levels of ASS1 and ASL might help regulate nitric oxide (NO) generation and mitigate its cytotoxic effects. Overall, our work adds to the understanding of urea cycle enzymes in a context-independent of ureagenesis, their role in ccRCC progression, and uncovers novel potential metabolic vulnerabilities in ccRCC.

Endogenous S-nitrosocysteine proteomic inventories identify a core of proteins in heart metabolic pathways.

Protein cysteine residues are essential for protein folding, participate in enzymatic catalysis, and coordinate the binding of metal ions to proteins. Enzymatically catalyzed and redox-dependent post-translational modifications of cysteine residues are also critical for signal transduction and regulation of protein function and localization. S-nitrosylation, the addition of a nitric oxide equivalent to a cysteine residue, is a redox-dependent modification. In this study, we curated and analyzed four different studies that employed various chemoselective platforms coupled to mass spectrometry to precisely identify S-nitrosocysteine residues in mouse heart proteins. Collectively 1974 S-nitrosocysteine residues in 761 proteins were identified and 33.4% were identified in two or more studies. A core of 75 S-nitrosocysteine residues in 44 proteins were identified in all four studies. Bioinformatic analysis of each study indicated a significant enrichment of mitochondrial proteins participating in metabolism. Regulatory proteins in glycolysis, TCA cycle, oxidative phosphorylation and ATP production, long chain fatty acid ß-oxidation, and ketone and amino acid metabolism constitute the major functional pathways impacted by protein S-nitrosylation. In the cardiovascular system, nitric oxide signaling regulates vasodilation and cardiac muscle contractility. The meta-analysis of the proteomic data supports the hypothesis that nitric oxide signaling via protein S-nitrosylation is also a regulator of cardiomyocyte metabolism that coordinates fuel utilization to maximize ATP production. As such, protein cysteine S-nitrosylation represents a third functional dimension of nitric oxide signaling in the cardiovascular system to ensure optimal cardiac function.

Organic mercury solid phase chemoselective capture for proteomic identification of S-nitrosated proteins and peptides.

Cysteine S-nitrosation mediates NO signaling and protein function under pathophysiological conditions. Herein, we provide a detailed protocol regarding the organic mercury chemoselective enrichment of S-nitrosated proteins and peptides. We discuss key aspects of the enrichment strategy and provide technical tips for the best performance of the experimental protocol.

Human Placental Transcriptome Reveals Critical Alterations in Inflammation and Energy Metabolism with Fetal Sex Differences in Spontaneous Preterm Birth.

A well-functioning placenta is crucial for normal gestation and regulates the nutrient, gas, and waste exchanges between the maternal and fetal circulations and is an important endocrine organ producing hormones that regulate both the maternal and fetal physiologies during pregnancy. Placental insufficiency is implicated in spontaneous preterm birth (SPTB). We proposed that deficits in the capacity of the placenta to maintain bioenergetic and metabolic stability during pregnancy may ultimately result in SPTB. To explore our hypothesis, we performed a RNA-seq study in male and female placentas from women with SPTB (<36 weeks gestation) compared to normal pregnancies (≥38 weeks gestation) to assess the alterations in the gene expression profiles. We focused exclusively on Black women (cases and controls), who are at the highest risk of SPTB. Six hundred and seventy differentially expressed genes were identified in male SPTB placentas. Among them, 313 and 357 transcripts were increased and decreased, respectively. In contrast, only 61 differentially expressed genes were identified in female SPTB placenta. The ingenuity pathway analysis showed alterations in the genes and canonical pathways critical for regulating inflammation, oxidative stress, detoxification, mitochondrial function, energy metabolism, and the extracellular matrix. Many upstream regulators and master regulators important for nutrient-sensing and metabolism were also altered in SPTB placentas, including the PI3K complex, TGFB1/SMADs, SMARCA4, TP63, CDKN2A, BRCA1, and NFAT. The transcriptome was integrated with published human placental metabolome to assess the interactions of altered genes and metabolites. Collectively, significant and biologically relevant alterations in the transcriptome were identified in SPTB placentas with fetal sex disparities. Altered energy metabolism, mitochondrial function, inflammation, and detoxification may underly the mechanisms of placental dysfunction in SPTB.

Multimodality assessment of heart failure with preserved ejection fraction skeletal muscle reveals differences in the machinery of energy fuel metabolism.

AIMS: Skeletal muscle (SkM) abnormalities may impact exercise capacity in patients with heart failure with preserved ejection fraction (HFpEF). We sought to quantify differences in SkM oxidative phosphorylation capacity (OxPhos), fibre composition, and the SkM proteome between HFpEF, hypertensive (HTN), and healthy participants. METHODS AND RESULTS: Fifty-nine subjects (20 healthy, 19 HTN, and 20 HFpEF) performed a maximal-effort cardiopulmonary exercise test to define peak oxygen consumption (VO2, peak ), ventilatory threshold (VT), and VO2 efficiency (ratio of total work performed to O2 consumed). SkM OxPhos was assessed using Creatine Chemical-Exchange Saturation Transfer (CrCEST, n = 51), which quantifies unphosphorylated Cr, before and after plantar flexion exercise. The half-time of Cr recovery (t1/2, Cr ) was taken as a metric of in vivo SkM OxPhos. In a subset of subjects (healthy = 13, HTN = 9, and HFpEF = 12), percutaneous biopsy of the vastus lateralis was performed for myofibre typing, mitochondrial morphology, and proteomic and phosphoproteomic analysis. HFpEF subjects demonstrated lower VO2,peak , VT, and VO2 efficiency than either control group (all P < 0.05). The t1/2, Cr was significantly longer in HFpEF (P = 0.005), indicative of impaired SkM OxPhos, and correlated with cycle ergometry exercise parameters. HFpEF SkM contained fewer Type I myofibres (P = 0.003). Proteomic analyses demonstrated (a) reduced levels of proteins related to OxPhos that correlated with exercise capacity and (b) reduced ERK signalling in HFpEF. CONCLUSIONS: Heart failure with preserved ejection fraction patients demonstrate impaired functional capacity and SkM OxPhos. Reductions in the proportions of Type I myofibres, proteins required for OxPhos, and altered phosphorylation signalling in the SkM may contribute to exercise intolerance in HFpEF.

TCA cycle metabolic compromise due to an aberrant S-nitrosoproteome in HIV-associated neurocognitive disorder with methamphetamine use.

In the brain, both HIV-1 and methamphetamine (meth) use result in increases in oxidative and nitrosative stress. This redox stress is thought to contribute to the pathogenesis of HIV-associated neurocognitive disorder (HAND) and further worsening cognitive activity in the setting of drug abuse. One consequence of such redox stress is aberrant protein S-nitrosylation, derived from nitric oxide, which may disrupt normal protein activity. Here, we report an improved, mass spectrometry-based technique to assess S-nitrosylated protein in human postmortem brains using selective enrichment of S-nitrosocysteine residues with an organomercury resin. The data show increasing S-nitrosylation of tricarboxylic acid (TCA) enzymes in the setting of HAND and HAND/meth use compared with HIV+ control brains without CNS pathology. The consequence is systematic inhibition of multiple TCA cycle enzymes, resulting in energy collapse that can contribute to the neuronal and synaptic damage observed in HAND and meth use.

Mitochondrial morphology, bioenergetics and proteomic responses in fatty acid oxidation disorders.

Mutations in nuclear genes encoding for mitochondrial proteins very long-chain acyl-CoA dehydrogenase (VLCAD) and trifunctional protein (TFP) cause rare autosomal recessive disorders. Studies in fibroblasts derived from patients with mutations in VLCAD and TFP exhibit mitochondrial defects. To gain insights on pathological changes that account for the mitochondrial deficits we performed quantitative proteomic, biochemical, and morphometric analyses in fibroblasts derived from subjects with three different VLCAD and three different TFP mutations. Proteomic data that was corroborated by antibody-based detection, indicated reduced levels of VLCAD and TFP protein in cells with VLCAD and TFP mutations respectively, which in part accounted for the diminished fatty acid oxidation capacity. Decreased mitochondrial respiratory capacity in cells with VLCAD and TFP mutations was quantified after glucose removal and cells with TFP mutations had lower levels of glycogen. Despite these energetic deficiencies, the cells with VLCAD and TFP mutations did not exhibit changes in mitochondria morphology, distribution, fusion and fission, quantified by either confocal or transmission electron microscopy and corroborated by proteomic and antibody-based protein analysis. Fibroblasts with VLCAD and to a lesser extend cells with TFP mutations had increased levels of mitochondrial respiratory chain proteins and proteins that facilitate the assembly of respiratory complexes. With the exception of reduced levels of catalase and glutathione S-transferase theta-1 in cells with TFP mutations, the levels of 45 proteins across all major intracellular antioxidant networks were similar between cells with VLCAD and TFP mutations and non-disease controls. Collectively the data indicate that despite the metabolic deficits, cells with VLCAD and TFP mutations maintain their proteomic integrity to preserve cellular and mitochondria architecture, support energy production and protect against oxidative stress.

The effect of dietary nitrate on exercise capacity in chronic kidney disease: a randomized controlled pilot study.

BACKGROUND: Chronic Kidney Disease (CKD) patients exhibit a reduced exercise capacity that impacts quality of life. Dietary nitrate supplementation has been shown to have favorable effects on exercise capacity in disease populations by reducing the oxygen cost of exercise. This study investigated whether dietary nitrates would acutely improve exercise capacity in CKD patients. METHODS AND RESULTS: In this randomized, double-blinded crossover study, 12 Stage 3-4 CKD patients (Mean ± SEM: Age, 60 ± 5yrs; eGFR, 50.3 ± 4.6 ml/min/1.73 m2) received an acute dose of 12.6 mmol of dietary nitrate in the form of concentrated beetroot juice (BRJ) and a nitrate depleted placebo (PLA). Skeletal muscle mitochondrial oxidative function was assessed using near-infrared spectroscopy. Cardiopulmonary exercise testing was performed on a cycle ergometer, with intensity increased by 25 W every 3 min until volitional fatigue. Plasma nitric oxide (NO) metabolites (NOm; nitrate, nitrite, low molecular weight S-nitrosothiols, and metal bound NO) were determined by gas-phase chemiluminescence. Plasma NOm values were significantly increased following BRJ (BRJ vs. PLA: 1074.4 ± 120.4 µM vs. 28.4 ± 6.6 µM, p < 0.001). Total work performed (44.4 ± 10.6 vs 39.6 ± 9.9 kJ, p = 0.03) and total exercise time (674 ± 85 vs 627 ± 86s, p = 0.04) were significantly greater following BRJ. Oxygen consumption at the ventilatory threshold was also improved by BRJ (0.90 ± 0.08 vs. 0.74 ± 0.06 L/min, p = 0.04). These changes occurred in the absence of improved skeletal muscle mitochondrial oxidative capacity (p = 0.52) and VO2peak (p = 0.35). CONCLUSIONS: Our findings demonstrate that inorganic nitrate can acutely improve exercise capacity in CKD patients. The effects of chronic nitrate supplementation on CKD related exercise intolerance should be investigated in future studies.

Systematic elucidation of neuron-astrocyte interaction in models of amyotrophic lateral sclerosis using multi-modal integrated bioinformatics workflow.

Cell-to-cell communications are critical determinants of pathophysiological phenotypes, but methodologies for their systematic elucidation are lacking. Herein, we propose an approach for the Systematic Elucidation and Assessment of Regulatory Cell-to-cell Interaction Networks (SEARCHIN) to identify ligand-mediated interactions between distinct cellular compartments. To test this approach, we selected a model of amyotrophic lateral sclerosis (ALS), in which astrocytes expressing mutant superoxide dismutase-1 (mutSOD1) kill wild-type motor neurons (MNs) by an unknown mechanism. Our integrative analysis that combines proteomics and regulatory network analysis infers the interaction between astrocyte-released amyloid precursor protein (APP) and death receptor-6 (DR6) on MNs as the top predicted ligand-receptor pair. The inferred deleterious role of APP and DR6 is confirmed in vitro in models of ALS. Moreover, the DR6 knockdown in MNs of transgenic mutSOD1 mice attenuates the ALS-like phenotype. Our results support the usefulness of integrative, systems biology approach to gain insights into complex neurobiological disease processes as in ALS and posit that the proposed methodology is not restricted to this biological context and could be used in a variety of other non-cell-autonomous communication mechanisms.

AMPA Receptor Surface Expression Is Regulated by S-Nitrosylation of Thorase and Transnitrosylation of NSF.

The regulation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) trafficking affects multiple brain functions, such as learning and memory. We have previously shown that Thorase plays an important role in the internalization of AMPARs from the synaptic membrane. Here, we show that N-methyl-d-aspartate receptor (NMDAR) activation leads to increased S-nitrosylation of Thorase and N-ethylmaleimide-sensitive factor (NSF). S-nitrosylation of Thorase stabilizes Thorase-AMPAR complexes and enhances the internalization of AMPAR and interaction with protein-interacting C kinase 1 (PICK1). S-nitrosylated NSF is dependent on the S-nitrosylation of Thorase via trans-nitrosylation, which modulates the surface insertion of AMPARs. In the presence of the S-nitrosylation-deficient C137L Thorase mutant, AMPAR trafficking, long-term potentiation, and long-term depression are impaired. Overall, our data suggest that both S-nitrosylation and interactions of Thorase and NSF/PICK1 are required to modulate AMPAR-mediated synaptic plasticity. This study provides critical information that elucidates the mechanism underlying Thorase and NSF-mediated trafficking of AMPAR complexes.