scholarly journals Inhibition of Lithium Sensitive Orai1/ STIM1 Expression and Store Operated Ca2+ Entry in Chorea-Acanthocytosis Neurons by NF-κB Inhibitor Wogonin

2018 ◽  
Vol 51 (1) ◽  
pp. 278-289 ◽  
Author(s):  
Basma Sukkar ◽  
Stefan Hauser ◽  
Lisann Pelzl ◽  
Zohreh Hosseinzadeh ◽  
Itishri Sahu ◽  
...  
Keyword(s):  
Cell Death ◽  
Cortical Neurons ◽  
Lithium Treatment ◽  
Cell Types ◽  
Protein Abundance ◽  
Loss Of Function ◽  
Atpase Inhibitor ◽  
Patch Clamp Recording ◽  
Transcript Levels ◽  
Induced Pluripotent

Background/Aims: The neurodegenerative disease Chorea-Acanthocytosis (ChAc) is caused by loss-of-function-mutations of the chorein-encoding gene VPS13A. In ChAc neurons transcript levels and protein abundance of Ca2+ release activated channel moiety (CRAC) Orai1 as well as its regulator STIM1/2 are decreased, resulting in blunted store operated Ca2+-entry (SOCE) and enhanced suicidal cell death. SOCE is up-regulated and cell death decreased by lithium. The effects of lithium are paralleled by upregulation of serum & glucocorticoid inducible kinase SGK1 and abrogated by pharmacological SGK1 inhibition. In other cell types SGK1 has been shown to be partially effective by upregulation of NFκB, a transcription factor stimulating the expression of Orai1 and STIM. The present study explored whether pharmacological inhibition of NFκB interferes with Orai1/STIM1/2 expression and SOCE and their upregulation by lithium in ChAc neurons. Methods: Cortical neurons were differentiated from induced pluripotent stem cells generated from fibroblasts of ChAc patients and healthy volunteers. Orai1 and STIM1 transcript levels and protein abundance were estimated from qRT-PCR and Western blotting, respectively, cytosolic Ca2+-activity ([Ca2+]i) from Fura-2-fluorescence, SOCE from increase of [Ca2+]i following Ca2+ re-addition after Ca2+-store depletion with sarco-endoplasmatic Ca2+-ATPase inhibitor thapsigargin (1µM), as well as CRAC current utilizing whole cell patch clamp recording. Results: Orai1 and STIM1 transcript levels and protein abundance as well as SOCE and CRAC current were significantly enhanced by lithium treatment (2 mM, 24 hours). These effects were reversed by NFκB inhibitor wogonin (50 µM). Conclusion: The stimulation of expression and function of Orai1/STIM1/2 by lithium in ChAc neurons are disrupted by pharmacological NFκB inhibition.

scholarly journals Genome-wide CRISPR/Cas9 screening in human iPS derived cardiomyocytes uncovers novel mediators of doxorubicin cardiotoxicity

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Valerie Sapp ◽  
Aitor Aguirre ◽  
Gayatri Mainkar ◽  
Jeffrey Ding ◽  
Eric Adler ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Types ◽  
Functional Screening ◽  
Loss Of Function ◽  
Ips Cell ◽  
Genome Wide ◽  
Doxorubicin Cardiotoxicity ◽  
Induced Pluripotent ◽  
Genome Scale ◽  
Human Specific

AbstractHuman induced pluripotent stem (iPS) cell technologies coupled with genetic engineering now facilitate the study of the molecular underpinnings of disease in relevant human cell types. Application of CRISPR/Cas9-based approaches for genome-scale functional screening in iPS-derived cells, however, has been limited by technical constraints, including inefficient transduction in pooled format, loss of library representation, and poor cellular differentiation. Herein, we present optimized approaches for whole-genome CRISPR/Cas9 based screening in human iPS derived cardiomyocytes with near genome-wide representation at both the iPS and differentiated cell stages. As proof-of-concept, we perform a screen to investigate mechanisms underlying doxorubicin mediated cell death in iPS derived cardiomyocytes. We identified two poorly characterized, human-specific transporters (SLCO1A2, SLCO1B3) whose loss of function protects against doxorubicin-cardiotoxicity, but does not affect cell death in cancer cells. This study provides a technical framework for genome-wide functional screening in iPS derived cells and identifies new targets to mitigate doxorubicin-cardiotoxicity in humans.

scholarly journals 1α,25(OH) 2D3 Sensitive Cytosolic pH Regulation and Glycolytic Flux in Human Endometrial Ishikawa Cells

2017 ◽  
Vol 41 (2) ◽  
pp. 678-688 ◽  
Author(s):  
Ni Zeng ◽  
Yuetao Zhou ◽  
Shaqiu Zhang ◽  
Yogesh Singh ◽  
Bing Shi ◽  
...  
Keyword(s):  
Cell Death ◽  
Transcription Factor ◽  
Lactate Concentration ◽  
B Cell Lymphoma ◽  
Transport Processes ◽  
Glycolytic Flux ◽  
Protein Abundance ◽  
Cytosolic Ph ◽  
Transcript Levels ◽  
Ishikawa Cells

Background/Aims: Tumor cell proliferation is modified by 1,25-Dihydroxy-Vitamin D3 (1,25(OH)2D3), a steroid hormone predominantly known for its role in calcium and phosphorus metabolism. Key properties of tumor cells include enhanced glycolytic flux with excessive consumption of glucose and formation of lactate. As glycolysis is highly sensitive to cytosolic pH, maintenance of glycolysis requires export of H+ ions and lactate, which is in part accomplished by Na+/H+ exchangers, such as NHE1 and monocarboxylate transporters, such as MCT4. An effect of 1,25(OH)2D3 on those transport processes has, however, never been reported. As cytosolic pH impacts on apoptosis, the study further explored the effect of 1,25(OH)2D3 on apoptosis and on the apoptosis regulating kinase AKT, transcription factor Forkhead box O-3 (FOXO3A) and B-cell lymphoma protein BCL-2. Methods: In human endometrial adenocarcinoma (Ishikawa) cells, cytosolic pH (pHi) was determined utilizing (2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein [BCECF] fluorescence, Na+/H+ exchanger activity from Na+ dependent realkalinization after an ammonium pulse, NHE1 and MCT4 transcript levels using qRT-PCR, NHE1, MCT4, total & phospho AKT, total & phospho-FOXO3A and BCL-2 protein abundance by Western blotting, lactate concentration in the supernatant utilizing a colorimetric enzyme assay and cell death quantification using CytoTox 96®, Annexin V and Propidium Iodide staining. Results: A 24 hours treatment with 1,25(OH)2D3 (100 nM) significantly increased cytosolic pH (pHi), significantly decreased Na+/H+ exchanger activity, NHE1 and MCT4 transcript levels as well as protein abundance and significantly increased lactate concentration in the supernatant. Treatment of Ishikawa cells with 1,25(OH)2D3 (100 nM) further triggered apoptosis, an effect paralleled by decreased phosphorylation of AKT and FOXO3A as well as decreased abundance of BCL-2. Conclusions: In Ishikawa cells 1,25(OH)2D3 is a powerful stimulator of glycolysis, an effect presumably due to cytosolic alkalinization. Despite stimulation of glycolysis, 1,25(OH)2D3 stimulates slightly but significantly suicidal cell death, an effect presumably in part due to decreased activation of AKT with decreased inhibition of pro-apoptotic transcription factor FOXO3A and downregulation of the anti-apoptotic protein BCL-2.

scholarly journals Loss of function of the mitochondrial peptidase PITRM1 induces proteotoxic stress and Alzheimer’s disease-like pathology in human cerebral organoids

2020 ◽  
Author(s):  
Dina Ivanyuk ◽  
María José Pérez ◽  
Vasiliki Panagiotakopoulou ◽  
Gabriele Di Napoli ◽  
Dario Brunetti ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Cell Death ◽  
Cortical Neurons ◽  
Amyloid Β ◽  
Neuronal Cell ◽  
Protein Aggregates ◽  
Neuronal Cultures ◽  
Loss Of Function ◽  
Proteotoxic Stress

AbstractMutations in pitrilysin metallopeptidase 1 (PITRM1), a mitochondrial protease involved in mitochondrial precursor processing and degradation, result in a slow-progressive syndrome, characterized by cerebellar ataxia, psychotic episodes and obsessive behavior as well as cognitive decline. To investigate the pathogenetic mechanisms of mitochondrial presequence processing, we employed cortical neurons and cerebral organoids generated from PITRM1 knockout human induced pluripotent stem cells (iPSCs). PITRM1 deficiency strongly induced mitochondrial unfolded protein response (UPRmt) and enhanced mitochondrial clearance in iPSC-derived neurons. Furthermore, we observed increased levels of amyloid precursor protein and amyloid β in PITRM1 knockout neurons. However, neither cell death nor protein aggregates were observed in 2D iPSC-derived cortical neuronal cultures. On the contrary, cerebral organoids generated from PITRM1 knockout iPSCs spontaneously developed over time pathological features of Alzheimer’s disease (AD), including accumulation of protein aggregates, tau pathology, and neuronal cell death. Importantly, we provide evidence for a protective role of UPRmt and mitochondrial clearance against impaired mitochondrial presequence processing and proteotoxic stress. In summary, we propose a novel concept of PITRM1-linked neurological syndrome whereby defects of mitochondrial presequence processing induce an early activation of UPRmt that, in turn, modulates cytosolic quality control pathways. Thus our work supports a mechanistic link between mitochondrial function and common neurodegenerative proteinopathies.

scholarly journals An in vitro bioengineered model of the human arterial neurovascular unit to study neurodegenerative diseases

2020 ◽  
Author(s):  
Jerome Robert ◽  
Nicholas Weilinger ◽  
Li-Ping Zao ◽  
Stefano Cataldi ◽  
Emily Button ◽  
...  
Keyword(s):  
Neurodegenerative Diseases ◽  
Cortical Neurons ◽  
Neurovascular Unit ◽  
Induced Pluripotent Stem Cell ◽  
Cell Types ◽  
Experimental Models ◽  
Flow Conditions ◽  
Anatomy And Physiology ◽  
Induced Pluripotent

Abstract Introduction: The neurovascular unit (NVU) – the interaction between the neurons and the cerebrovasculature – is increasingly important to interrogate through human-based experimental models. Although advanced models of cerebral capillaries have been developed in the last decade, there is currently noin vitro3-dimensional (3D) perfusible model of the human cortical arterial NVU. Method: We used a tissue-engineering technique to develop a scaffold-directed, perfusible, 3D human NVU that is cultured in native-like flow conditions that mimics the anatomy and physiology of cortical penetrating arteries. Results: This system, composed of primary human vascular cells (endothelial cells, smooth muscle cells and astrocytes) and induced pluripotent stem cell (iPSC) derived neurons, demonstrates a physiological multilayer organization of the involved cell types. It also reproduces key characteristics of cortical neurons and astrocytes, as well as the formation of a selective and functional endothelial barrier. We further provide proof-of-principle that our in vitro human arterial NVU may be suitable to study neurodegenerative diseases such as Alzheimer’s disease (AD), as we report both phosphorylated tau and beta-amyloid accumulation in our model over time. Finally, we show that our arterial NVU model enables the study of neuronal and glial fluid biomarkers. Conclusion: This model is a suitable tool to investigate arterial NVU functions such as neuronal electrophysiology in health and disease. Further the design of platform allows culture under native-like flow conditions for extended periods of time and yields sufficient tissue and media for downstream immunohistochemistry and biochemistry analyses.

scholarly journals An in vitro bioengineered model of the human arterial neurovascular unit to study neurodegenerative diseases

2020 ◽  
Author(s):  
Jerome Robert ◽  
Nicholas Weilinger ◽  
Li-Ping Zao ◽  
Stefano Cataldi ◽  
Emily Button ◽  
...  
Keyword(s):  
Neurodegenerative Diseases ◽  
Cortical Neurons ◽  
Neurovascular Unit ◽  
Induced Pluripotent Stem Cell ◽  
Cell Types ◽  
Experimental Models ◽  
Flow Conditions ◽  
Anatomy And Physiology ◽  
Induced Pluripotent

Abstract Introduction: The neurovascular unit (NVU) – the interaction between the neurons and the cerebrovasculature – is increasingly important to interrogate through human-based experimental models. Although advanced models of cerebral capillaries have been developed in the last decade, there is currently no in vitro 3-dimensional (3D) perfusible model of the human cortical arterial NVU.Method: We used a tissue-engineering technique to develop a scaffold-directed, perfusible, 3D human NVU that is cultured in native-like flow conditions that mimics the anatomy and physiology of cortical penetrating arteries.Results: This system, composed of primary human vascular cells (endothelial cells, smooth muscle cells and astrocytes) and induced pluripotent stem cell (iPSC) derived neurons, demonstrates a physiological multilayer organization of the involved cell types. It reproduces key characteristics of cortical neurons and astrocytes and enables formation of a selective and functional endothelial barrier. We provide proof-of-principle data showing that this in vitro human arterial NVU may be suitable to study neurovascular components of neurodegenerative diseases such as Alzheimer’s disease (AD), as endogenously produced phosphorylated tau and beta-amyloid accumulate in the model over time. Finally, neuronal and glial fluid biomarkers relevant to neurodegenerative diseases are measurable in our arterial NVU model.Conclusion: This model is a suitable research tool to investigate arterial NVU functions in healthy and disease states. Further, the design of the platform allows culture under native-like flow conditions for extended periods of time and yields sufficient tissue and media for downstream immunohistochemistry and biochemistry analyses.

scholarly journals Reduction of the neuroprotective transcription factor Npas4 results in increased neuronal necrosis, inflammation and brain lesion size following ischaemia

2015 ◽  
Vol 36 (8) ◽  
pp. 1449-1463 ◽  
Author(s):  
Fong Chan Choy ◽  
Thomas S Klarić ◽  
Wai Khay Leong ◽  
Simon A Koblar ◽  
Martin D Lewis
Keyword(s):  
Cell Death ◽  
Transcription Factor ◽  
Cortical Neurons ◽  
Neuroprotective Effect ◽  
Critical Role ◽  
Brain Lesion ◽  
Lesion Size ◽  
Loss Of Function ◽  
Neuronal Necrosis ◽  
Ischaemic Brain

Stroke is the second leading cause of death and the most frequent cause of adult disability. Neuronal Per-Arnt-Sim domain protein 4 (Npas4) is an activity-dependent transcription factor whose expression is induced in various brain insults, including cerebral ischaemia. Although previous studies have demonstrated that Npas4 plays a critical role in protecting neurons against neurodegenerative insults, the neuroprotective effect of Npas4 in response to ischaemic brain injury remains unknown. In this study, we used a loss-of-function approach to examine the neuroprotective potential of Npas4 in the context of ischaemic damage. Using oxygen and glucose deprivation, we demonstrated that the knockdown of Npas4 in mouse cortical neurons resulted in increased susceptibility to cell death. The protective effect of Npas4 was further investigated in vivo using a photochemically-induced stroke model in mice. We found a significantly larger lesion size and increased neurodegeneration in Npas4 knockout mice as compared to wild-type mice. Moreover, we also showed that ablation of Npas4 caused an increase in activated astrocytes and microglia, pro-inflammatory cytokines interleukin-6 and tumour necrosis factor alpha levels and a switch from apoptotic to necrotic cell death. Taken together, these data suggest that Npas4 plays a neuroprotective role in ischaemic stroke by limiting progressive neurodegeneration and neuroinflammation.

scholarly journals Genome-wide CRISPR/Cas9-knockout in human induced Pluripotent Stem Cell (iPSC)-derived macrophages

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Elena Navarro-Guerrero ◽  
Chwen Tay ◽  
Justin P. Whalley ◽  
Sally A. Cowley ◽  
Ben Davies ◽  
...  
Keyword(s):  
Cancer Progression ◽  
Genome Engineering ◽  
Induced Pluripotent Stem Cell ◽  
Cell Types ◽  
Loss Of Function ◽  
Lentiviral Transduction ◽  
Genome Wide ◽  
A Genome ◽  
Human Pathology ◽  
Induced Pluripotent

AbstractGenome engineering using CRISPR/Cas9 technology enables simple, efficient and precise genomic modifications in human cells. Conventional immortalized cell lines can be easily edited or screened using genome-wide libraries with lentiviral transduction. However, cell types derived from the differentiation of induced Pluripotent Stem Cells (iPSC), which often represent more relevant, patient-derived models for human pathology, are much more difficult to engineer as CRISPR/Cas9 delivery to these differentiated cells can be inefficient and toxic. Here, we present an efficient, lentiviral transduction protocol for delivery of CRISPR/Cas9 to macrophages derived from human iPSC with efficiencies close to 100%. We demonstrate CRISPR/Cas9 knockouts for three nonessential proof-of-concept genes—HPRT1, PPIB and CDK4. We then scale the protocol and validate for a genome-wide pooled CRISPR/Cas9 loss-of-function screen. This methodology enables, for the first time, systematic exploration of macrophage involvement in immune responses, chronic inflammation, neurodegenerative diseases and cancer progression, using efficient genome editing techniques.

scholarly journals An in vitro bioengineered model of the human arterial neurovascular unit to study neurodegenerative diseases

2020 ◽  
Vol 15 (1) ◽  
Author(s):  
Jerome Robert ◽  
Nicholas L. Weilinger ◽  
Li-Ping Cao ◽  
Stefano Cataldi ◽  
Emily B. Button ◽  
...  
Keyword(s):  
Neurodegenerative Diseases ◽  
Cortical Neurons ◽  
Neurovascular Unit ◽  
Induced Pluripotent Stem Cell ◽  
Cell Types ◽  
Experimental Models ◽  
Flow Conditions ◽  
Anatomy And Physiology ◽  
Induced Pluripotent

Abstract Introduction The neurovascular unit (NVU) – the interaction between the neurons and the cerebrovasculature – is increasingly important to interrogate through human-based experimental models. Although advanced models of cerebral capillaries have been developed in the last decade, there is currently no in vitro 3-dimensional (3D) perfusible model of the human cortical arterial NVU. Method We used a tissue-engineering technique to develop a scaffold-directed, perfusible, 3D human NVU that is cultured in native-like flow conditions that mimics the anatomy and physiology of cortical penetrating arteries. Results This system, composed of primary human vascular cells (endothelial cells, smooth muscle cells and astrocytes) and induced pluripotent stem cell (iPSC) derived neurons, demonstrates a physiological multilayer organization of the involved cell types. It reproduces key characteristics of cortical neurons and astrocytes and enables formation of a selective and functional endothelial barrier. We provide proof-of-principle data showing that this in vitro human arterial NVU may be suitable to study neurovascular components of neurodegenerative diseases such as Alzheimer’s disease (AD), as endogenously produced phosphorylated tau and beta-amyloid accumulate in the model over time. Finally, neuronal and glial fluid biomarkers relevant to neurodegenerative diseases are measurable in our arterial NVU model. Conclusion This model is a suitable research tool to investigate arterial NVU functions in healthy and disease states. Further, the design of the platform allows culture under native-like flow conditions for extended periods of time and yields sufficient tissue and media for downstream immunohistochemistry and biochemistry analyses.

scholarly journals Phenotypic variation within and across transcriptomic cell types in mouse motor cortex

2020 ◽  
Author(s):  
Federico Scala ◽  
Dmitry Kobak ◽  
Matteo Bernabucci ◽  
Yves Bernaerts ◽  
Cathryn René Cadwell ◽  
...  
Keyword(s):  
Motor Cortex ◽  
Cortical Neurons ◽  
Adult Mouse ◽  
Cell Types ◽  
Neuronal Diversity ◽  
Patch Clamp Recording ◽  
Electrophysiological Properties ◽  
Single Cell Rna Sequencing ◽  
Electric Space ◽  
Almost All

Cortical neurons exhibit astounding diversity in gene expression as well as in morphological and electrophysiological properties. Most existing neural taxonomies are based on either transcriptomic or morpho-electric criteria, as it has been technically challenging to study both aspects of neuronal diversity in the same set of cells. Here we used Patch-seq to combine patch-clamp recording, biocytin staining, and single-cell RNA sequencing of over 1300 neurons in adult mouse motor cortex, providing a comprehensive morpho-electric annotation of almost all transcriptomically defined neural cell types. We found that, although broad families of transcriptomic types (Vip, Pvalb, Sst, etc.) had distinct and essentially non-overlapping morpho-electric phenotypes, individual transcriptomic types within the same family were not well-separated in the morpho-electric space. Instead, there was a continuum of variability in morphology and electrophysiology, with neighbouring transcriptomic cell types showing similar morpho-electric features, often without clear boundaries between them. Our results suggest that neural types in the neocortex do not always form discrete entities. Instead, neurons follow a hierarchy consisting of distinct non-overlapping branches at the level of families, but can form continuous and correlated transcriptomic and morpho-electrical landscapes within families.

scholarly journals Vacuolar processing enzymes, AmVPE1 and AmVPE2, as potential executors of ethylene regulated programmed cell death in the lace plant (Aponogeton madagascariensis)

Botany ◽  
2018 ◽  
Vol 96 (4) ◽  
pp. 235-247 ◽  
Author(s):  
Gaolathe Rantong ◽  
Arunika H.L.A.N. Gunawardena
Keyword(s):  
Cell Death ◽  
Programmed Cell Death ◽  
Leaf Development ◽  
Cell Types ◽  
Ethylene Biosynthesis ◽  
Activity Levels ◽  
Developmentally Regulated ◽  
Transcript Levels ◽  
Processing Enzymes ◽  
Laser Capture Microscopy

Perforation formation in Aponogeton madagascariensis (Mirb.) H.Bruggen (lace plant) is an excellent model for studying developmentally regulated programmed cell death (PCD). In this study, we isolated and identified two lace plant vacuolar processing enzymes (VPEs) and investigated their involvement in PCD and throughout leaf development. Lace plant VPE transcript levels were determined during seven different stages of leaf development. PCD and non-PCD cells from “window” stage leaves (in which perforations are forming) were separated through laser-capture microscopy and their transcript levels were also determined. VPE activity was also studied between the cell types, through a VPE activity-based probe JOPD1. Additionally, VPE transcript levels were studied in plants treated with an ethylene biosynthesis inhibitor, aminoethoxyvinylglycine (AVG). The two isolated VPEs, AmVPE1 and AmVPE2, are vegetative type VPEs. AmVPE1 had higher transcript levels during a pre-perforation developmental stage, immediately prior to visible signs of PCD. AmVPE2 transcript levels were higher later during window and late window stages. Both VPEs had higher transcript and activity levels in PCD compared with the non-PCD cells. AVG treatment inhibited PCD and associated increases in VPE transcript levels. Our results suggested that VPEs are involved in the execution of the ethylene-related PCD in the lace plant.

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