scholarly journals Presynaptic dysfunction in CASK-related neurodevelopmental disorders

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Martin Becker ◽  
Francesca Mastropasqua ◽  
Jan Philipp Reising ◽  
Simon Maier ◽  
Mai-Lan Ho ◽  
...  
Keyword(s):  
Induced Pluripotent Stem Cell ◽  
Splice Site Mutation ◽  
Autism Spectrum ◽  
Resonance Spectroscopy ◽  
Limited Information ◽  
Site Mutation ◽  
Mutation Carriers ◽  
Human Neurons

Abstract CASK-related disorders are genetically defined neurodevelopmental syndromes. There is limited information about the effects of CASK mutations in human neurons. Therefore, we sought to delineate CASK-mutation consequences and neuronal effects using induced pluripotent stem cell-derived neurons from two mutation carriers. One male case with autism spectrum disorder carried a novel splice-site mutation and a female case with intellectual disability carried an intragenic tandem duplication. We show reduction of CASK protein in maturing neurons from the mutation carriers, which leads to significant downregulation of genes involved in presynaptic development and of CASK protein interactors. Furthermore, CASK-deficient neurons showed decreased inhibitory presynapse size as indicated by VGAT staining, which may alter the excitatory–inhibitory (E/I) balance in developing neural circuitries. Using in vivo magnetic resonance spectroscopy quantification of GABA in the male mutation carrier, we further highlight the possibility to validate in vitro cellular data in the brain. Our data show that future pharmacological and clinical studies on targeting presynapses and E/I imbalance could lead to specific treatments for CASK-related disorders.

scholarly journals Presynaptic dysfunction in CASK-related neurodevelopmental disorders

2019 ◽  
Author(s):  
Martin Becker ◽  
Francesca Mastropasqua ◽  
Jan Philipp Reising ◽  
Simon Maier ◽  
Mai-Lan Ho ◽  
...  
Keyword(s):  
Induced Pluripotent Stem Cell ◽  
Resonance Spectroscopy ◽  
Limited Information ◽  
Mutation Carriers ◽  
Gene Sets ◽  
Human Neurons ◽  
Induced Pluripotent ◽  
Human Neuronal Cells

SummaryCASK-related disorders are a genetically defined group of neurodevelopmental syndromes. There is limited information about the effects of CASK mutations in human neurons. Therefore, we sought to delineate CASK mutation consequences and neuronal level effects using induced pluripotent stem cell-derived neurons from two mutation carriers; one male diagnosed with ASD and a female with MICPCH. We show a reduction of the CASK protein in maturing neurons from the mutation carriers, which leads to significant downregulation of gene sets involved in presynaptic development and CASK protein interactors. Furthermore, CASK-deficient neurons showed decreased inhibitory presynapse size as indicated by VGAT staining, which may alter the excitatory-inhibitory (E/I) balance in developing neural circuitries. Using in vivo magnetic resonance spectroscopy quantification of GABA in the male mutation carrier, we further highlight the possibility to validate in vitro cellular data in brain. Our data shows that future pharmacological and clinical studies on targeting presynapses and E/I imbalance could lead to specific treatments for CASK-related disorders.HighlightsModelling of CASK-related disorders using iPSC-derived human neuronal cellsCASK mutations cause dysregulation of its protein interactor partnersReduced CASK levels primarily affect inhibitory presynapse developmentIn vitro GABAergic phenotype predicts in vivo neurotransmitter levels

scholarly journals Species-specific maturation profiles of human, chimpanzee and bonobo neural cells

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Maria C Marchetto ◽  
Branka Hrvoj-Mihic ◽  
Bilal E Kerman ◽  
Diana X Yu ◽  
Krishna C Vadodaria ◽  
...  
Keyword(s):  
Neural Progenitor Cells ◽  
Pyramidal Neurons ◽  
Induced Pluripotent Stem Cell ◽  
Neural Cells ◽  
Important Species ◽  
Comparative Analyses ◽  
Human Neurons ◽  
Species Specific

Comparative analyses of neuronal phenotypes in closely related species can shed light on neuronal changes occurring during evolution. The study of post-mortem brains of nonhuman primates (NHPs) has been limited and often does not recapitulate important species-specific developmental hallmarks. We utilize induced pluripotent stem cell (iPSC) technology to investigate the development of cortical pyramidal neurons following migration and maturation of cells grafted in the developing mouse cortex. Our results show differential migration patterns in human neural progenitor cells compared to those of chimpanzees and bonobos both in vitro and in vivo, suggesting heterochronic changes in human neurons. The strategy proposed here lays the groundwork for further comparative analyses between humans and NHPs and opens new avenues for understanding the differences in the neural underpinnings of cognition and neurological disease susceptibility between species.

In vitro Lipolysis as a Tool for the Establishment of IVIVC for Lipid-Based Drug Delivery Systems

2019 ◽  
Vol 16 (8) ◽  
pp. 688-697
Author(s):  
Ravinder Verma ◽  
Deepak Kaushik
Keyword(s):  
Drug Delivery ◽  
Ph Value ◽  
Electron Paramagnetic Resonance Spectroscopy ◽  
Rapid Screening ◽  
Resonance Spectroscopy ◽  
Fed State ◽  
In Vitro Lipolysis ◽  
Ph Stat

: In vitro lipolysis has emerged as a powerful tool in the development of in vitro in vivo correlation for Lipid-based Drug Delivery System (LbDDS). In vitro lipolysis possesses the ability to mimic the assimilation of LbDDS in the human biological system. The digestion medium for in vitro lipolysis commonly contains an aqueous buffer media, bile salts, phospholipids and sodium chloride. The concentrations of these compounds are defined by the physiological conditions prevailing in the fasted or fed state. The pH of the medium is monitored by a pH-sensitive electrode connected to a computercontrolled pH-stat device capable of maintaining a predefined pH value via titration with sodium hydroxide. Copenhagen, Monash and Jerusalem are used as different models for in vitro lipolysis studies. The most common approach used in evaluating the kinetics of lipolysis of emulsion-based encapsulation systems is the pH-stat titration technique. This is widely used in both the nutritional and the pharmacological research fields as a rapid screening tool. Analytical tools for the assessment of in vitro lipolysis include HPLC, GC, HPTLC, SEM, Cryo TEM, Electron paramagnetic resonance spectroscopy, Raman spectroscopy and Nanoparticle Tracking Analysis (NTA) for the characterization of the lipids and colloidal phases after digestion of lipids. Various researches have been carried out for the establishment of IVIVC by using in vitro lipolysis models. The current publication also presents an updated review of various researches in the field of in vitro lipolysis.

scholarly journals A predictive in vitro risk assessment platform for pro-arrhythmic toxicity using human 3D cardiac microtissues

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Celinda M. Kofron ◽  
Tae Yun Kim ◽  
Fabiola Munarin ◽  
Arvin H. Soepriatna ◽  
Rajeev J. Kant ◽  
...  
Keyword(s):  
Risk Assessment ◽  
Action Potentials ◽  
Induced Pluripotent Stem Cell ◽  
Human Action ◽  
Environmental Toxicants ◽  
Pharmaceutical Drugs ◽  
Industrial Chemicals ◽  
Endocrine Disrupting Chemical

AbstractCardiotoxicity of pharmaceutical drugs, industrial chemicals, and environmental toxicants can be severe, even life threatening, which necessitates a thorough evaluation of the human response to chemical compounds. Predicting risks for arrhythmia and sudden cardiac death accurately is critical for defining safety profiles. Currently available approaches have limitations including a focus on single select ion channels, the use of non-human species in vitro and in vivo, and limited direct physiological translation. We have advanced the robustness and reproducibility of in vitro platforms for assessing pro-arrhythmic cardiotoxicity using human induced pluripotent stem cell-derived cardiomyocytes and human cardiac fibroblasts in 3-dimensional microtissues. Using automated algorithms and statistical analyses of eight comprehensive evaluation metrics of cardiac action potentials, we demonstrate that tissue-engineered human cardiac microtissues respond appropriately to physiological stimuli and effectively differentiate between high-risk and low-risk compounds exhibiting blockade of the hERG channel (E4031 and ranolazine, respectively). Further, we show that the environmental endocrine disrupting chemical bisphenol-A (BPA) causes acute and sensitive disruption of human action potentials in the nanomolar range. Thus, this novel human 3D in vitro pro-arrhythmic risk assessment platform addresses critical needs in cardiotoxicity testing for both environmental and pharmaceutical compounds and can be leveraged to establish safe human exposure levels.

scholarly journals Mechanistic physiology-based pharmacokinetic modeling to elucidate vincristine-induced peripheral neuropathy following treatment with novel kinase inhibitors

2021 ◽  
Author(s):  
Venkatesh Pilla Reddy ◽  
Adrian J. Fretland ◽  
Diansong Zhou ◽  
Shringi Sharma ◽  
Buyun Chen ◽  
...  
Keyword(s):  
Peripheral Neuropathy ◽  
Kinase Inhibitors ◽  
Pbpk Model ◽  
Model Development ◽  
Limited Information ◽  
Time Curve ◽  
Btk Inhibitor ◽  
Chop Therapy

Abstract Purpose Limited information is available regarding the drug–drug interaction (DDI) potential of molecular targeted agents and rituximab plus cyclophosphamide, doxorubicin (hydroxydaunorubicin), vincristine (Oncovin), and prednisone (R-CHOP) therapy. The addition of the Bruton tyrosine kinase (BTK) inhibitor ibrutinib to R-CHOP therapy results in increased toxicity versus R-CHOP alone, including higher incidence of peripheral neuropathy. Vincristine is a substrate of P-glycoprotein (P-gp, ABCB1); drugs that inhibit P-gp could potentially cause increased toxicity when co-administered with vincristine through DDI. While the combination of the BTK inhibitor acalabrutinib and R-CHOP is being explored clinically, the DDI potential between these therapies is unknown. Methods A human mechanistic physiology-based pharmacokinetic (PBPK) model of vincristine following intravenous dosing was developed to predict potential DDI interactions with combination therapy. In vitro absorption, distribution, metabolism, and excretion and in vivo clinical PK parameters informed PBPK model development, which was verified by comparing simulated vincristine concentrations with observed clinical data. Results While simulations suggested no DDI between vincristine and ibrutinib or acalabrutinib in plasma, simulated vincristine exposure in muscle tissue was increased in the presence of ibrutinib but not acalabrutinib. Extrapolation of the vincristine mechanistic PBPK model to other P-gp substrates further suggested DDI risk when ibrutinib (area under the concentration–time curve [AUC] ratio: 1.8), but not acalabrutinib (AUC ratio: 0.92), was given orally with venetoclax or digoxin. Conclusion Overall, these data suggest low DDI risk between acalabrutinib and P-gp substrates with negligible increase in the potential risk of vincristine-induced peripheral neuropathy when acalabrutinib is added to R-CHOP therapy.

scholarly journals Region-specific elevations of glutamate + glutamine correlate with the sensory symptoms of autism spectrum disorders

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Jason L. He ◽  
Georg Oeltzschner ◽  
Mark Mikkelsen ◽  
Alyssa Deronda ◽  
Ashley D. Harris ◽  
...  
Keyword(s):  
Autism Spectrum Disorders ◽  
Strong Support ◽  
Empirical Support ◽  
Brain Regions ◽  
Autism Spectrum ◽  
Resonance Spectroscopy ◽  
Primary Sensorimotor Cortex ◽  
Spectrum Disorders ◽  
Inhibitory Signaling

AbstractIndividuals on the autism spectrum are often reported as being hyper- and/or hyporeactive to sensory input. These sensory symptoms were one of the key observations that led to the development of the altered excitation-inhibition (E-I) model of autism, which posits that an increase ratio of excitatory to inhibitory signaling may explain certain phenotypical expressions of autism spectrum disorders (ASD). While there has been strong support for the altered E-I model of autism, much of the evidence has come from animal models. With regard to in-vivo human studies, evidence for altered E-I balance in ASD come from studies adopting magnetic resonance spectroscopy (MRS). Spectral-edited MRS can be used to provide measures of the levels of GABA + (GABA + macromolecules) and Glx (glutamate + glutamine) in specific brain regions as proxy markers of inhibition and excitation respectively. In the current study, we found region-specific elevations of Glx in the primary sensorimotor cortex (SM1) in ASD. There were no group differences of GABA+ in either the SM1 or thalamus. Higher levels of Glx were associated with more parent reported difficulties of sensory hyper- and hyporeactivity, as well as reduced feed-forward inhibition during tactile perception in children with ASD. Critically, the finding of elevated Glx provides strong empirical support for increased excitation in ASD. Our results also provide a clear link between Glx and the sensory symptoms of ASD at both behavioral and perceptual levels.

scholarly journals Persistence of intramyocardially transplanted murine induced pluripotent stem cell-derived cardiomyocytes from different developmental stages

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Gabriel Peinkofer ◽  
Martina Maass ◽  
Kurt Pfannkuche ◽  
Agapios Sachinidis ◽  
Stephan Baldus ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Stem Cell ◽  
Cell Adhesion ◽  
Developmental Stage ◽  
Pluripotent Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
Induced Pluripotent

Abstract Background Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) are regarded as promising cell type for cardiac cell replacement therapy, but it is not known whether the developmental stage influences their persistence and functional integration in the host tissue, which are crucial for a long-term therapeutic benefit. To investigate this, we first tested the cell adhesion capability of murine iPSC-CM in vitro at three different time points during the differentiation process and then examined cell persistence and quality of electrical integration in the infarcted myocardium in vivo. Methods To test cell adhesion capabilities in vitro, iPSC-CM were seeded on fibronectin-coated cell culture dishes and decellularized ventricular extracellular matrix (ECM) scaffolds. After fixed periods of time, stably attached cells were quantified. For in vivo experiments, murine iPSC-CM expressing enhanced green fluorescent protein was injected into infarcted hearts of adult mice. After 6–7 days, viable ventricular tissue slices were prepared to enable action potential (AP) recordings in transplanted iPSC-CM and surrounding host cardiomyocytes. Afterwards, slices were lysed, and genomic DNA was prepared, which was then used for quantitative real-time PCR to evaluate grafted iPSC-CM count. Results The in vitro results indicated differences in cell adhesion capabilities between day 14, day 16, and day 18 iPSC-CM with day 14 iPSC-CM showing the largest number of attached cells on ECM scaffolds. After intramyocardial injection, day 14 iPSC-CM showed a significant higher cell count compared to day 16 iPSC-CM. AP measurements revealed no significant difference in the quality of electrical integration and only minor differences in AP properties between d14 and d16 iPSC-CM. Conclusion The results of the present study demonstrate that the developmental stage at the time of transplantation is crucial for the persistence of transplanted iPSC-CM. iPSC-CM at day 14 of differentiation showed the highest persistence after transplantation in vivo, which may be explained by a higher capability to adhere to the extracellular matrix.

Abstract 518: In Vitro and in vivo Disease Modeling Using Patient Derived iPSCs to Characterize the Calcification Phenotype in Arterial Calcification Due to Deficiency of CD73

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Cynthia St. Hilaire ◽  
Hui Jin ◽  
Yuting Huang ◽  
Dan Yang ◽  
Alejandra Negro ◽  
...  
Keyword(s):  
Vascular Calcification ◽  
Disease Modeling ◽  
Induced Pluripotent Stem Cell ◽  
Dermal Fibroblasts ◽  
In Vivo Studies ◽  
Arterial Calcification ◽  
Patient Specific ◽  
And Control

Objective: The objective of this study was to develop a patient-specific induced pluripotent stem cell (iPSC)-based disease model to understand the process by which CD73-deficiency leads to vascular calcification in the disease, Arterial Calcification due to Deficiency of CD73 (ACDC). Approach & Results: ACDC is an autosomal recessive disease resulting from mutations in the gene encoding for CD73, which converts extracellular AMP to adenosine. CD73-deficiency manifests with tortuosity and vascular calcification of the medial layer of lower-extremity arteries, a pathology associated with diabetes and chronic kidney disease. We previously identified that dermal fibroblasts isolated from ACDC patients calcify in vitro, however in vivo studies of the vasculature are limited, as murine models of CD73 deficiency do not recapitulate the human disease phenotype. Thus, we created iPSCs from ACDC patients and control fibroblasts. ACDC and Control iPSCs form teratomas when injected in immune-compromised mice, however ACDC iPSC teratomas exhibit extensive calcifications. Control and ACDC iPSCs were differentiated down the mesenchymal lineage (MSC) and while there was no difference in chondrogenesis and adipogenesis, ACDC iMSCs underwent osteogenesis sooner than control iPSC, have higher activity of tissue-nonspecific alkaline phosphatase (TNAP), and lower levels of extracellular adenosine. During osteogenic simulation, TNAP activity in ACDC cells significantly increased adenosine levels, however, not to levels needed for functional compensatory stimulation of the adenosine receptors. Inhibition of TNAP with levimisole ablates this increase in adenosine. Treatment with an A2b adenosine receptor (AR) agonist drastically reduced TNAP activity in vitro, and calcification in ACDC teratomas, as did treatment with etidronate, which is currently being tested in a clinical trial on ACDC patients. Conclusions: These results illustrate a pro-osteogenic phenotype in CD73-deficient cells whereby TNAP activity attempts to compensate for CD73 deficiency, but subsequently induces calcification that can be reversed by activation of the A2bAR. The iPSC teratoma model may be used to screen other potential therapeutics for calcification disorders.

Effects of hypercapnia on ECoG and oxidative metabolism in neonatal dog brain

1995 ◽  
Vol 78 (6) ◽  
pp. 2272-2278 ◽  
Author(s):  
H. Yoshioka ◽  
H. Miyake ◽  
D. S. Smith ◽  
B. Chance ◽  
T. Sawada ◽  
...  
Keyword(s):  
Electrical Activity ◽  
Resonance Spectroscopy ◽  
Respiratory Enzymes ◽  
Arterial Pco2 ◽  
A Value ◽  
Electrocorticogram Ecog ◽  
The Brain ◽  
Mitochondrial Respiratory

The effects of hypercapnia on cerebral electrical activity and mitochondrial oxidative phosphorylation were studied in the anesthetized neonatal dog by using the electrocorticogram (ECoG) and 31P-magnetic resonance spectroscopy. Three levels of hypercapnia with arterial PCO2 values of approximately 70, 100, and 140 Torr reduced the intracellular pH of the brain from 7.11 to 6.99, 6.87, and 6.76, respectively. These levels of hypercapnia also reduced ADP concentration ([ADP]) from 21.5 to 18.1, 14.8, and 12.9 microM as well as the average ECoG power output by 20, 30, and 40%. A Michaelis-Menten relationship for the mitochondrial respiratory enzymes was fitted with [ADP] and the change in the average ECoG. The result suggests that mitochondrial respiration is regulated by [ADP] and that the in vivo Michaelis-Menten constant for ADP was 21 microM, a value close to the in vitro value. The mitochondrial maximal reaction velocity was reduced by only 10% during hypercapnia and showed no relationship with the degree of acidosis, suggesting that mitochondrial respiratory enzymes are not responsible for the inhibition of the brain electrical activity.

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