Glutamate-Glutamine Homeostasis is Perturbed in Neurons and Astrocytes derived from Patient iPSC Models of Frontotemporal Dementia

Abstract Frontotemporal dementia (FTD) is amongst the most prevalent early onset dementias and even though it is clinically, pathologically and genetically heterogeneous, a crucial involvement of metabolic perturbations in FTD pathology is being recognized. However, changes in metabolism at the cellular level, implicated in FTD and in neurodegeneration in general, are still poorly understood. Here we generate induced human pluripotent stem cells (hiPSCs) from patients carrying mutations in CHMP2B (FTD3) and isogenic controls generated via CRISPR/Cas9 gene editing with subsequent neuronal and glial differentiation and characterization. FTD3 neurons show a dysregulation of glutamate-glutamine related metabolic pathways mapped by 13C-labelling coupled to mass spectrometry. FTD3 astrocytes show increased uptake of glutamate whilst glutamate metabolism is largely maintained. Using quantitative proteomics and live-cell metabolic analyses, we elucidate molecular determinants and functional alterations of neuronal and glial energy metabolism in FTD3. Importantly, correction of the mutations rescues such pathological phenotypes. Notably, these findings implicate dysregulation of key enzymes crucial for glutamate-glutamine homeostasis in FTD3 pathogenesis which may underlie vulnerability to neurodegeneration.

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Neuronal Glutamate-Glutamine Homeostasis is Perturbed in a Patient-Derived iPSC Model of Frontotemporal Dementia

10.21203/rs.2.23638/v1 ◽

2020 ◽

Author(s):

Blanca Irene Aldana ◽

Yu Zhang ◽

Pia Jensen ◽

Sofie Kjellerup Christensen ◽

Troels Tolstrup Nielsen ◽

...

Keyword(s):

Mass Spectrometry ◽

Stem Cells ◽

Frontotemporal Dementia ◽

Pluripotent Stem Cells ◽

Metabolic Pathways ◽

Quantitative Proteomics ◽

Gene Editing ◽

Cellular Level ◽

Molecular Determinants ◽

Neuronal Energy Metabolism

Abstract Frontotemporal dementia (FTD) is amongst the most prevalent early onset dementias and even though it is clinically, pathologically and genetically heterogeneous, a crucial involvement of metabolic perturbations in FTD pathology is being recognized. However, changes in metabolism at the cellular level, implicated in FTD and in neurodegeneration in general, are still poorly understood. Here we generate induced human pluripotent stem cells (hiPSCs) from patients carrying mutations in CHMP2B (FTD3) and isogenic controls generated via CRISPR/Cas9 gene editing with subsequent neuronal differentiation and characterization. FTD3 neurons show a dysregulation of glutamate-glutamine related metabolic pathways mapped by 13 C-labelling coupled to mass spectrometry. Using quantitative proteomics and seahorse analyses, we elucidate molecular determinants and functional alterations of neuronal energy metabolism in FTD3. Importantly, correction of the mutations rescues such pathological phenotypes. Notably, these findings implicate dysregulation of key enzymes crucial for glutamate-glutamine homeostasis in FTD3 pathogenesis which may underlie vulnerability to neurodegeneration.

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Glutamate-glutamine homeostasis is perturbed in neurons and astrocytes derived from patient iPSC models of frontotemporal dementia

Molecular Brain ◽

10.1186/s13041-020-00658-6 ◽

2020 ◽

Vol 13 (1) ◽

Cited By ~ 1

Author(s):

Blanca I. Aldana ◽

Yu Zhang ◽

Pia Jensen ◽

Abinaya Chandrasekaran ◽

Sofie K. Christensen ◽

...

Keyword(s):

Mass Spectrometry ◽

Stem Cells ◽

Energy Metabolism ◽

Frontotemporal Dementia ◽

Pluripotent Stem Cells ◽

Quantitative Proteomics ◽

Tca Cycle ◽

Glutamate Metabolism ◽

The Tca Cycle ◽

Molecular Determinants

Abstract Frontotemporal dementia (FTD) is amongst the most prevalent early onset dementias and even though it is clinically, pathologically and genetically heterogeneous, a crucial involvement of metabolic perturbations in FTD pathology is being recognized. However, changes in metabolism at the cellular level, implicated in FTD and in neurodegeneration in general, are still poorly understood. Here we generate induced human pluripotent stem cells (hiPSCs) from patients carrying mutations in CHMP2B (FTD3) and isogenic controls generated via CRISPR/Cas9 gene editing with subsequent neuronal and glial differentiation and characterization. FTD3 neurons show a dysregulation of glutamate-glutamine related metabolic pathways mapped by 13C-labelling coupled to mass spectrometry. FTD3 astrocytes show increased uptake of glutamate whilst glutamate metabolism is largely maintained. Using quantitative proteomics and live-cell metabolic analyses, we elucidate molecular determinants and functional alterations of neuronal and glial energy metabolism in FTD3. Importantly, correction of the mutations rescues such pathological phenotypes. Notably, these findings implicate dysregulation of key enzymes crucial for glutamate-glutamine homeostasis in FTD3 pathogenesis which may underlie vulnerability to neurodegeneration. Graphical abstract Neurons derived from human induced pluripotent stem cells (hiPSCs) of patients carrying mutations in CHMP2B (FTD3) display major metabolic alterations compared to CRISPR/Cas9 generated isogenic controls. Using quantitative proteomics, 13C-labelling coupled to mass spectrometry metabolic mapping and seahorse analyses, molecular determinants and functional alterations of neuronal and astrocytic energy metabolism in FTD3 were characterized. Our findings implicate dysregulation of glutamate-glutamine homeostasis in FTD3 pathogenesis. In addition, FTD3 neurons recapitulate glucose hypometabolism observed in FTD patient brains. The impaired mitochondria function found here is concordant with disturbed TCA cycle activity and decreased glycolysis in FTD3 neurons. FTD3 neuronal glutamine hypermetabolism is associated with up-regulation of PAG expression and, possibly, ROS production. Distinct compartments of glutamate metabolism can be suggested for the FTD3 neurons. Endogenous glutamate generated from glutamine via PAG may enter the TCA cycle via AAT (left side of neuron) while exogenous glutamate taken up from the extracellular space may be incorporated into the TCA cycle via GDH (right side of the neuron) FTD3 astrocytic glutamate uptake is upregulated whilst glutamate metabolism is largely maintained. Finally, pharmacological reversal of glutamate hypometabolism manifesting from decreased GDH expression should be explored as a novel therapeutic intervention for treating FTD3.

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Characteristics of serum metabolites in sporadic amyotrophic lateral sclerosis patients based on gas chromatography-mass spectrometry

Scientific Reports ◽

10.1038/s41598-021-00312-8 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Rui Jia ◽

Qiaoyi Chen ◽

Qingqing Zhou ◽

Ronghua Zhang ◽

Jiaoting Jin ◽

...

Keyword(s):

Mass Spectrometry ◽

Gas Chromatography ◽

Amyotrophic Lateral Sclerosis ◽

Metabolic Pathways ◽

Gas Chromatography Mass Spectrometry ◽

Glutamate Metabolism ◽

Serum Metabolites ◽

Beta Alanine ◽

Als Patients ◽

Lateral Sclerosis

AbstractTo identify differential metabolites and metabolic pathways and provide guidance for the novel biomarkers for diagnosis and prognosis of amyotrophic lateral sclerosis (ALS). ALS patients and people without nervous diseases were recruited. Metabolomic analysis was performed using gas chromatography-mass spectrometry (GC/MS). The orthogonal projections to latent structures discriminant analysis (OPLS-DA) were used to identify differential metabolites. Kyoto Encyclopedia of Genes and Genomes and MetaboAnalyst were used to identify metabolic pathways. 75 metabolites were detected and aligned. The OPLS-DA showed the metabolomic profile of ALS patients and those in the fast-progression and slow-progression ALS groups differed from that of CTRL (p < 0.05). The levels of maltose, glyceric acid, lactic acid, beta-alanine, phosphoric acid, glutamic acid, ethanolamine and glycine in ALS were significantly higher, while 2,4,6-tri-tert-butylbenzenethiol was lower. Glycine, serine and threonine metabolism, D-glutamine and D-glutamate metabolism, alanine, aspartate, and glutamate metabolism, beta-alanine metabolism, and pyruvate metabolism were significantly altered metabolic pathways in ALS. ROC was used to discriminate ALS from CTRL with an AUC of 0.898 (p < 0.001) using 2,4,6-tri-tert-butylbenzenethiol, beta-alanine, glycine, and ethanolamine. The serum metabolites and metabolic pathways in ALS patients are significantly altered compared with CTRL. These findings may contribute to the early diagnosis of ALS.

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Mass Spectrometry-based Label-free Quantitative Proteomics To Study the Effect of 3PO Drug at Cellular Level

ACS Medicinal Chemistry Letters ◽

10.1021/acsmedchemlett.8b00593 ◽

2019 ◽

Vol 10 (4) ◽

pp. 577-583 ◽

Cited By ~ 3

Author(s):

Sarath Babu Nukala ◽

Giovanna Baron ◽

Giancarlo Aldini ◽

Marina Carini ◽

Alfonsina D’Amato

Keyword(s):

Mass Spectrometry ◽

Quantitative Proteomics ◽

Cellular Level ◽

Label Free

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Ultra-Performance Liquid Chromatography-Q-Exactive Orbitrap-Mass Spectrometry Analysis for Metabolic Communication between Heart and Kidney in Adriamycin-Induced Nephropathy Rats

Kidney & Blood Pressure Research ◽

10.1159/000519015 ◽

2021 ◽

pp. 1-12

Author(s):

Chunliu Wang ◽

Jiping Liang ◽

Wenwen Yang ◽

Shixiang Wang ◽

Jie Yu ◽

...

Keyword(s):

Mass Spectrometry ◽

Liquid Chromatography ◽

Metabolic Pathways ◽

Ultra Performance Liquid Chromatography ◽

Glutamate Metabolism ◽

Model Group ◽

Inflammatory Infiltration ◽

Orbitrap Mass Spectrometry ◽

Q Exactive ◽

Heart Blood

Background/Aims: Although the adriamycin-induced nephropathy model is frequently employed in the study of nephrotic syndrome and focal segmental glomerulosclerosis, the accompanying myocardial damage has always been a cause for concern. Therefore, there is a great need to study cardiorenal communication in this model. Methods: An adriamycin-induced nephropathy model was established via tail vein injection. The levels of the biochemical indicators serum albumin, serum globulin, serum total protein, serum cholesterol, serum creatinine (SCr), urinary protein, and urinary creatinine (UCr) were measured, and histopathological changes in the heart and kidneys were assessed using hematoxylin-eosin staining. Metabolomic changes in the heart, blood, and kidneys were analyzed using the metabolomics method based on ultra-performance liquid chromatography Q-Exactive Orbitrap mass spectrometry. Results: Compared with the control group, the model group showed significant decreases in serum protein and total protein levels, albumin/globulin ratio, and creatinine clearance rate as well as significant increases in serum cholesterol, SCr, urinary protein, and UCr levels. Significant pathological changes were observed in the renal pathology sections in the model group, including diffusely merged glomerular epithelial cells, inflammatory infiltration, and vacuolated glomerular cells. Additionally, thickened myocardial fibers, swollen nuclei, inflammatory infiltration, and partial myocardial necrosis could be seen in the cardiac pathology sections in the model group. Based on multivariate statistical analysis, a total of 20 differential metabolites associated with 15 metabolic pathways were identified in the heart, 7 differential metabolites with 7 metabolic pathways were identified in the blood, and 16 differential metabolites with 21 metabolic pathways were identified in the kidney. Moreover, 6 common metabolic pathways shared by the heart and kidney were identified: arginine and proline metabolism; arginine biosynthesis; glutathione metabolism; alanine, aspartate, and glutamate metabolism; beta-alanine metabolism; and histidine metabolism. Among these metabolic pathways, alanine, aspartate, and glutamate metabolism was shared by the heart, blood, and kidney. Succinic acid was found to be the key regulatory metabolite in cardiorenal metabolic communication. Conclusion: Six metabolic pathways were found to be involved in cardiorenal metabolic communication in an adriamycin-induced nephropathy model, in which alanine, aspartate, and glutamate metabolism may be the metabolic link between the heart and kidney in the development and maintenance of oxidative stress and inflammation. Succinic acid may serve as a key regulatory metabolic switch or marker of cardiac and renal co-injury, as shown in an adriamycin-induced nephropathy model.

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Development of Algorithms for Mass Spectrometry-based Label-free Quantitative Proteomics*

PROGRESS IN BIOCHEMISTRY AND BIOPHYSICS ◽

10.3724/sp.j.1206.2010.00560 ◽

2011 ◽

Vol 38 (6) ◽

pp. 506-518 ◽

Cited By ~ 2

Author(s):

Wei ZHANG ◽

Ji-Yang ZHANG ◽

Hui LIU ◽

Han-Chang SUN ◽

Chang-Ming XU ◽

...

Keyword(s):

Mass Spectrometry ◽

Quantitative Proteomics ◽

Label Free

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Inductively Coupled Plasma Mass Spectrometry (ICP-MS) Applications in Quantitative Proteomics

Protein and Peptide Letters ◽

10.2174/0929866523666161213094936 ◽

2017 ◽

Vol 24 (3) ◽

pp. 253-266 ◽

Cited By ~ 9

Author(s):

Osama Chahrour ◽

John Malone

Keyword(s):

Mass Spectrometry ◽

Quantitative Proteomics ◽

Inductively Coupled Plasma ◽

Inductively Coupled ◽

Icp Ms ◽

Plasma Mass Spectrometry

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Mass Spectrometry Techniques: Principles and Practices for Quantitative Proteomics

Current Protein and Peptide Science ◽

10.2174/1389203721666200921153513 ◽

2020 ◽

Vol 21 ◽

Author(s):

Rocco J. Rotello ◽

Timothy D. Veenstra

Keyword(s):

Mass Spectrometry ◽

Quantitative Proteomics ◽

Protein Identification ◽

Dynamic Nature ◽

Complete Coverage ◽

The Past ◽

Proteome Level ◽

A Cell ◽

Quantitative Changes ◽

Made In

: In the current omics-age of research, major developments have been made in technologies that attempt to survey the entire repertoire of genes, transcripts, proteins, and metabolites present within a cell. While genomics has led to a dramatic increase in our understanding of such things as disease morphology and how organisms respond to medications, it is critical to obtain information at the proteome level since proteins carry out most of the functions within the cell. The primary tool for obtaining proteome-wide information on proteins within the cell is mass spectrometry (MS). While it has historically been associated with the protein identification, developments over the past couple of decades have made MS a robust technology for protein quantitation as well. Identifying quantitative changes in proteomes is complicated by its dynamic nature and the inability of any technique to guarantee complete coverage of every protein within a proteome sample. Fortunately, the combined development of sample preparation and MS methods have made it capable to quantitatively compare many thousands of proteins obtained from cells and organisms.

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T9. EPIGENETIC PROFILING IN SCHIZOPHRENIA DERIVED HUMAN INDUCED PLURIPOTENT STEM CELLS (HIPSCS) AND NEURONS

Schizophrenia Bulletin ◽

10.1093/schbul/sbaa029.569 ◽

2020 ◽

Vol 46 (Supplement_1) ◽

pp. S234-S234

Author(s):

Lorna Farrelly ◽

Shuangping Zhang ◽

Erin Flaherty ◽

Aaron Topol ◽

Nadine Schrode ◽

...

Keyword(s):

Gene Expression ◽

Mass Spectrometry ◽

Stem Cells ◽

Induced Pluripotent Stem Cells ◽

Pluripotent Stem Cells ◽

Neural Progenitor Cells ◽

Gene Expression Pattern ◽

Early Gene ◽

Label Free ◽

Induced Pluripotent

Abstract Background Schizophrenia (SCZ) is a severe psychiatric disorder affecting ~1% of the world’s population. It is largely heritable with genetic risk reflected by a combination of common variants of small effect and highly penetrant rare mutations. Chromatin modifications are known to play critical roles in the mediation of many neurodevelopmental processes, and, when disturbed, may also contribute to the precipitation of psychiatric disorders, such as SCZ. While a handful of candidate-based studies have measured changes in promoter-bound histone modifications, few mechanistic studies have been carried out to explore how these modifications may affect chromatin to precipitate behavioral phenotypes associated with the disease. Methods We applied an unbiased proteomics approach to evaluate the epigenetic landscape of SCZ in human induced pluripotent stem cells (hiPSC), neural progenitor cells (NPCs) and neurons from SCZ patients vs. matched controls. We utilized proteomics-based, label free liquid chromatography mass spectrometry (LC-MS/MS) on purified histones from these cells and confirmed our results by western blotting in postmortem SCZ cortical brain tissues. Furthermore we validated our findings with the application of histone interaction assays and structural and biophysical assessments to identify and confirm novel chromatin ‘readers’. To relate our findings to a SCZ phenotype we used a SCZ rodent model of prepulse inhibition (PPI) to perform pharmacological manipulations and behavioral assessments. Results Using label free mass spectrometry we performed PTM screening of hiPSCs, NPCs and matured neurons derived from SCZ patients and matched controls. We identified, amongst others, altered patterns of hyperacetylation in SCZ neurons. Additionally we identified enhanced binding of particular acetylation ‘reader’ proteins. Pharmacological inhibition of such proteins in an animal model of amphetamine sensitization ameliorated PPI deficits further validating this epigenetic signature in SCZ. Discussion Recent evidence indicates that relevance and patterns of acetylation in epigenetics advances beyond its role in transcription and small molecule inhibitors of these aberrant interactions hold promise as useful therapeutics. This study identifies a role for modulating gene expression changes associated with a SCZ epigenetic signature and warrants further investigation in terms of how this early gene expression pattern perhaps determines susceptibility or severity of the SCZ disease trajectory.

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