scholarly journals Tau interactome mapping reveals dynamic processes in synapses and mitochondria associated with neurodegenerative disease

2021 ◽  
Author(s):  
Tara E Tracy ◽  
Jesus Madero-Perez ◽  
Danielle Swaney ◽  
Timothy S Chang ◽  
Michelle Moritz ◽  
...  
Keyword(s):  
Affinity Purification ◽  
Induced Pluripotent Stem Cell ◽  
Proximity Ligation Assay ◽  
Spatiotemporal Resolution ◽  
Proximity Ligation ◽  
Cytosolic Domains ◽  
Presynaptic Vesicle ◽  
Underlying Mechanisms ◽  
Induced Pluripotent ◽  
Affinity Purification Mass Spectrometry

Tau (MAPT) drives neuronal dysfunction in Alzheimer's disease (AD) and other tauopathies. To dissect the underlying mechanisms, we combined an engineered ascorbic acid peroxidase (APEX) approach with quantitative affinity purification mass spectrometry (AP-MS) followed by proximity ligation assay (PLA) to characterize Tau interactomes modified by neuronal activity and mutations that cause frontotemporal dementia (FTD) in human induced pluripotent stem cell (iPSC)-derived neurons. We established activity-dependent interactions of Tau with presynaptic vesicle proteins during Tau secretion and mapped the exact APEX-tau-induced biotinylated tyrosines to the cytosolic domains of the interacting vesicular proteins. We showed that FTD mutations impair bioenergetics and markedly diminished Tau's interaction with mitochondria proteins, which were downregulated in AD brains of multiple cohorts and correlated with disease severity. These multi-modal and dynamic Tau interactomes with unprecedented spatiotemporal resolution shed novel insights into Tau's role in neuronal function and disease-related processes with potential therapeutic targets to block Tau-mediated pathogenesis.

scholarly journals Mitochondria Endoplasmic Reticulum Contact Sites (MERCs): Proximity Ligation Assay as a Tool to Study Organelle Interaction

2021 ◽  
Vol 9 ◽  
Author(s):  
Sara Benhammouda ◽  
Anjali Vishwakarma ◽  
Priya Gatti ◽  
Marc Germain
Keyword(s):  
Endoplasmic Reticulum ◽  
Fluorescence Probes ◽  
Proximity Ligation Assay ◽  
Proximity Ligation ◽  
Membrane Contact Sites ◽  
Contact Sites ◽  
Membrane Contact ◽  
Endogenous Proteins ◽  
Underlying Mechanisms

Organelles cooperate with each other to regulate vital cellular homoeostatic functions. This occurs through the formation of close connections through membrane contact sites. Mitochondria-Endoplasmic-Reticulum (ER) contact sites (MERCS) are one of such contact sites that regulate numerous biological processes by controlling calcium and metabolic homeostasis. However, the extent to which contact sites shape cellular biology and the underlying mechanisms remain to be fully elucidated. A number of biochemical and imaging approaches have been established to address these questions, resulting in the identification of a number of molecular tethers between mitochondria and the ER. Among these techniques, fluorescence-based imaging is widely used, including analysing signal overlap between two organelles and more selective techniques such as in-situ proximity ligation assay (PLA). While these two techniques allow the detection of endogenous proteins, preventing some problems associated with techniques relying on overexpression (FRET, split fluorescence probes), they come with their own issues. In addition, proper image analysis is required to minimise potential artefacts associated with these methods. In this review, we discuss the protocols and outline the limitations of fluorescence-based approaches used to assess MERCs using endogenous proteins.

scholarly journals A Mechanism Leading to Changes in Copy Number Variations Affected by Transcriptional Level Might Be Involved in Evolution, Embryonic Development, Senescence, and Oncogenesis Mediated by Retrotransposons

2021 ◽  
Vol 9 ◽  
Author(s):  
Yunpeng Sui ◽  
Shuanghong Peng
Keyword(s):  
Embryonic Development ◽  
Copy Number ◽  
Three Dimensional ◽  
Induced Pluripotent Stem Cell ◽  
Copy Number Variations ◽  
Mrna Splicing ◽  
Transcriptional Level ◽  
Underlying Mechanisms ◽  
Induced Pluripotent ◽  
Hiv 1

In recent years, more and more evidence has emerged showing that changes in copy number variations (CNVs) correlated with the transcriptional level can be found during evolution, embryonic development, and oncogenesis. However, the underlying mechanisms remain largely unknown. The success of the induced pluripotent stem cell suggests that genome changes could bring about transformations in protein expression and cell status; conversely, genome alterations generated during embryonic development and senescence might also be the result of genome changes. With rapid developments in science and technology, evidence of changes in the genome affected by transcriptional level has gradually been revealed, and a rational and concrete explanation is needed. Given the preference of the HIV-1 genome to insert into transposons of genes with high transcriptional levels, we propose a mechanism based on retrotransposons facilitated by specific pre-mRNA splicing style and homologous recombination (HR) to explain changes in CNVs in the genome. This mechanism is similar to that of the group II intron that originated much earlier. Under this proposed mechanism, CNVs on genome are dynamically and spontaneously extended in a manner that is positively correlated with transcriptional level or contract as the cell divides during evolution, embryonic development, senescence, and oncogenesis, propelling alterations in them. Besides, this mechanism explains several critical puzzles in these processes. From evidence collected to date, it can be deduced that the message contained in genome is not just three-dimensional but will become four-dimensional, carrying more genetic information.

Abstract 520: Structural, Molecular, and Electrophysiological Maturation of Human Induced Pluripotent Stem Cell Derived Atrial Cardiomyocytes to Serve as a Model for Atrial Fibrillation

2020 ◽  
Vol 127 (Suppl_1) ◽  
Author(s):  
Olivia T Ly ◽  
Grace Brown ◽  
Liang HONG ◽  
Arvind Sridhar ◽  
Meihong Zhang ◽  
...  
Keyword(s):  
Atrial Fibrillation ◽  
Electrode Array ◽  
Induced Pluripotent Stem Cell ◽  
Rna Seq ◽  
Patch Clamping ◽  
Cellular Mechanisms ◽  
Transmission Electron ◽  
Underlying Mechanisms ◽  
Induced Pluripotent ◽  
Scn5a Mutation

Background: Atrial fibrillation (AF), the most common arrhythmia, is associated with significant morbidity and increased mortality. Although antiarrhythmic drugs are still commonly used to treat symptomatic AF, membrane-active drugs are incompletely and unpredictably effective, failing to target the underlying mechanisms of AF. Our pilot data shows atrial iPSC-CMs generated from a familial AF kindred recapitulated the electrophysiologic (EP) phenotype of an AF-linked SCN5A mutation, serving as a novel platform to target underlying cellular AF mechanisms. However, structural, molecular, and EP immaturity as compared to adult atrial CMs has hindered successful mechanistic evaluation. Objective: We aim to determine optimal condition(s) to enhance the maturity of atrial iPSC-CMs, establishing them as a novel platform to model AF, elucidate the cellular mechanisms, and identify/assess novel, mechanism based therapies. Methods: Maturity of atrial iPSC-CMs was enhanced using TID (T3, IGF-1, dexamethasone), fatty acids (FA), acute electrical stimulation (ES), and extracellular matrix (ECM) modulation. We examined for improvements in structural maturity (immunofluorescence, transmission electron microscopy), molecular (qPCR, RNA seq) and EP (patch clamping, multi-electrode array, high-throughput automated patch clamping). The maturity of atrial iPSC-CMs was compared with adult atrial CMs obtained from the same patient during cardiac surgery. Results and Conclusion: Fig. 1 demonstrates that acute ES, combined with TID and FA supplementation, significantly improves structural and in part EP maturity of atrial iPSC-CMs.

scholarly journals Cell Transplantation for Spinal Cord Injury: Tumorigenicity of Induced Pluripotent Stem Cell-Derived Neural Stem/Progenitor Cells

2018 ◽  
Vol 2018 ◽  
pp. 1-7 ◽  
Author(s):  
Junhao Deng ◽  
Yiling Zhang ◽  
Yong Xie ◽  
Licheng Zhang ◽  
Peifu Tang
Keyword(s):  
Spinal Cord ◽  
Stem Cells ◽  
Spinal Cord Injury ◽  
Embryonic Stem ◽  
Induced Pluripotent Stem Cell ◽  
Therapeutic Effects ◽  
Cord Injury ◽  
Neural Stem Progenitor Cells ◽  
Underlying Mechanisms ◽  
Induced Pluripotent

Spinal cord injury (SCI) is an intractable and worldwide difficult medical challenge with limited treatments. Neural stem/progenitor cell (NS/PC) transplantation derived from fetal tissues or embryonic stem cells (ESCs) has demonstrated therapeutic effects via replacement of lost neurons and severed axons and creation of permissive microenvironment to promote repair of spinal cord and axon regeneration but causes ethnical concerns and immunological rejections as well. Thus, the implementation of induced pluripotent stem cells (iPSCs), which can be generated from adult somatic cells and differentiated into NS/PCs, provides an effective alternation in the treatment of SCI. However, as researches further deepen, there is accumulating evidence that the use of iPSC-derived NS/PCs shows mounting concerns of safety, especially the tumorigenicity. This review discusses the tumorigenicity of iPSC-derived NS/PCs focusing on the two different routes of tumorigenicity (teratomas and true tumors) and underlying mechanisms behind them, as well as possible solutions to circumvent them.

scholarly journals Impaired p53-mediated DNA damage response contributes to microcephaly in Nijmegen Breakage Syndrome patient-derived cerebral organoids

2020 ◽  
Author(s):  
Soraia Martins ◽  
Lars Erichsen ◽  
Angeliki Datsi ◽  
Wasco Wruck ◽  
Wolfgang Goering ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Genetic Disorder ◽  
Clinical Manifestations ◽  
Induced Pluripotent Stem Cell ◽  
Nijmegen Breakage Syndrome ◽  
Damage Repair ◽  
Damage Response ◽  
Underlying Mechanisms ◽  
Induced Pluripotent

Nijmegen Breakage Syndrome (NBS) is a rare autosomal recessive genetic disorder caused by mutations within NBN, a DNA-damage repair protein. Hallmarks of NBS include several clinical manifestations such growth retardation, chromosomal instability, immunodeficiency and progressive microcephaly. However, the etiology of microcephaly in NBS patients remains elusive. Here, we employed induced pluripotent stem cell-derived brain organoids from two NBS patients to analyze the underlying mechanisms of microcephaly. We show that NBS-organoids carrying the homozygous 647del5 NBN mutation are significantly smaller in size with disrupted cyto-architecture Patient-derived organoids exhibit premature differentiation together with neuronatin (NNAT) overexpression and key pathways related to DNA damage response and cell cycle are differentially regulated compared to controls. Moreover, we show that after exposure to bleomycin, NBS organoids undergo a delayed p53-mediated DNA damage response and aberrant trans-synaptic signalling, which ultimately leads to neuronal apoptosis. Our data provide insights into how mutations within NBN alters neurogenesis in NBS patients, thus providing a proof of concept that cerebral organoids are a valuable tool for studying DNA damage-related disorders.

scholarly journals Mechanisms and Insights for the Development of Heart Failure Associated with Cancer Therapy

Children ◽  
2021 ◽  
Vol 8 (9) ◽  
pp. 829
Author(s):  
Claire Fraley ◽  
Sarah A. Milgrom ◽  
Lavanya Kondapalli ◽  
Matthew R. G. Taylor ◽  
Luisa Mestroni ◽  
...  
Keyword(s):  
Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
Childhood Cancer Survivors ◽  
Cardiovascular Complications ◽  
Cancer Therapies ◽  
Hematopoietic Stem ◽  
Targeted Cancer Therapies ◽  
Underlying Mechanisms ◽  
Induced Pluripotent

Cardiotoxicity is a well-recognized late effect among childhood cancer survivors. With various pediatric cancers becoming increasingly curable, it is imperative to understand the disease burdens that survivors may face in the future. In order to prevent or mitigate cardiovascular complications, we must first understand the mechanistic underpinnings. This review will examine the underlying mechanisms of cardiotoxicity that arise from traditional antineoplastic chemotherapies, radiation therapy, hematopoietic stem cell transplantation, as well as newer cellular therapies and targeted cancer therapies. We will then propose areas for prevention, primarily drawing from the anthracycline-induced cardiotoxicity literature. Finally, we will explore the role of human induced pluripotent stem cell cardiomyocytes and genetics in advancing the field of cardio-oncology.

scholarly journals Cell-type specific impact of glucocorticoid receptor activation on the developing brain

2020 ◽  
Author(s):  
Cristiana Cruceanu ◽  
Leander Dony ◽  
Anthi C. Krontira ◽  
David S. Fischer ◽  
Simone Roeh ◽  
...  
Keyword(s):  
Glucocorticoid Receptor ◽  
Induced Pluripotent Stem Cell ◽  
Receptor Activation ◽  
Cell Type ◽  
Specific Effects ◽  
Cell Type Specific ◽  
Underlying Mechanisms ◽  
Induced Pluripotent ◽  
Specific Impact

AbstractA fine-tuned balance of glucocorticoid receptor (GR) activation is essential for organ formation, with disturbances influencing health outcomes. Excess GR-activation in utero has been linked to brain-related negative outcomes, with unclear underlying mechanisms, especially regarding cell-type specific effects. To address this, we used an in vitro model of fetal human brain, induced pluripotent-stem-cell-derived cerebral organoids, and mapped GR-activation effects using single-cell transcriptomics across development. Interestingly, neurons showed targeted regulation of differentiation- and maturation-related transcripts, suggesting a delay of these processes upon GR-activation. Uniquely in neurons, differentially-expressed transcripts were significantly enriched for genes associated with behavior-related phenotypes and disorders. This suggests that aberrant GR-activation could impact proper neuronal maturation, leading to increased disease susceptibility, through neurodevelopmental processes at the interface of genetic susceptibility and environmental exposure.

scholarly journals Synaptotagmin-7 deficiency induces mania-like behavioral abnormalities through attenuating GluN2B activity

2020 ◽  
Vol 117 (49) ◽  
pp. 31438-31447
Author(s):  
Qiu-Wen Wang ◽  
Si-Yao Lu ◽  
Yao-Nan Liu ◽  
Yun Chen ◽  
Hui Wei ◽  
...  
Keyword(s):  
Hippocampal Neurons ◽  
Bipolar Depression ◽  
Glutamate Release ◽  
Induced Pluripotent Stem Cell ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Behavioral Abnormalities ◽  
Synaptic Region ◽  
Underlying Mechanisms ◽  
Induced Pluripotent

Synaptotagmin-7 (Syt7) probably plays an important role in bipolar-like behavioral abnormalities in mice; however, the underlying mechanisms for this have remained elusive. Unlike antidepressants that cause mood overcorrection in bipolar depression,N-methyl-d-aspartate receptor (NMDAR)-targeted drugs show moderate clinical efficacy, for unexplained reasons. Here we identified Syt7 single nucleotide polymorphisms (SNPs) in patients with bipolar disorder and demonstrated that mice lacking Syt7 or expressing the SNPs showed GluN2B-NMDAR dysfunction, leading to antidepressant behavioral consequences and avoidance of overcorrection by NMDAR antagonists. In human induced pluripotent stem cell (iPSC)-derived and mouse hippocampal neurons, Syt7 and GluN2B-NMDARs were localized to the peripheral synaptic region, and Syt7 triggered multiple forms of glutamate release to efficiently activate the juxtaposed GluN2B-NMDARs. Thus, while Syt7 deficiency and SNPs induced GluN2B-NMDAR dysfunction in mice, patient iPSC-derived neurons showed Syt7 deficit-induced GluN2B-NMDAR hypoactivity that was rescued by Syt7 overexpression. Therefore, Syt7 deficits induced mania-like behaviors in mice by attenuating GluN2B activity, which enabled NMDAR antagonists to avoid mood overcorrection.

scholarly journals APOE2 mitigates disease-related phenotypes in an isogenic hiPSC-based model of Alzheimer’s disease

2021 ◽  
Author(s):  
Nicholas Brookhouser ◽  
Sreedevi Raman ◽  
Carlye Frisch ◽  
Gayathri Srinivasan ◽  
David A. Brafman
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Association Studies ◽  
Induced Pluripotent Stem Cell ◽  
Genome Wide Association Studies ◽  
Protective Function ◽  
Model Of Alzheimer’S Disease ◽  
Neural Cultures ◽  
Underlying Mechanisms ◽  
Induced Pluripotent

AbstractGenome-wide association studies (GWAS) have identified polymorphism in the Apolipoprotein E gene (APOE) to be the most prominent risk factor for Alzheimer’s disease (AD). Compared to individuals homozygous for the APOE3 variant, individuals with the APOE4 variant have a significantly elevated risk of AD. On the other hand, longitudinal studies have shown that the presence of the APOE2 variant reduces the lifetime risk of developing AD by 40 percent. While there has been significant research that has identified the risk-inducing effects of APOE4, the underlying mechanisms by which APOE2 influences AD onset and progression have not been extensively explored. In this study, we utilize an isogenic human induced pluripotent stem cell (hiPSC)-based system to demonstrate that conversion of APOE3 to APOE2 greatly reduced the production of amyloid-beta (Aβ) peptides in hiPSC-derived neural cultures. Mechanistically, analysis of pure populations of neurons and astrocytes derived from these neural cultures revealed that mitigating effects of APOE2 are mediated by cell autonomous and non-autonomous effects. In particular, we demonstrated the reduction in Aβ is potentially driven by a mechanism related to non-amyloidogenic processing of amyloid precursor protein (APP), suggesting a gain of the protective function of the APOE2 variant. Together, this study provides insights into the risk-modifying effects associated with the APOE2 allele and establishes a platform to probe the mechanisms by which APOE2 enhances neuroprotection against AD.

scholarly journals Proximity Ligation Assay Detection of Protein–DNA Interactions—Is There a Link between Heme Oxygenase-1 and G-quadruplexes?

Antioxidants ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 94
Author(s):  
Wojciech Krzeptowski ◽  
Patryk Chudy ◽  
Grzegorz Sokołowski ◽  
Monika Żukowska ◽  
Anna Kusienicka ◽  
...  
Keyword(s):  
Stem Cells ◽  
Heme Oxygenase ◽  
Direct Interaction ◽  
Proximity Ligation Assay ◽  
Limiting Factor ◽  
Heme Oxygenase 1 ◽  
Hematopoietic Stem ◽  
Proximity Ligation ◽  
Rare Cells ◽  
Induced Pluripotent

G-quadruplexes (G4) are stacked nucleic acid structures that are stabilized by heme. In cells, they affect DNA replication and gene transcription. They are unwound by several helicases but the composition of the repair complex and its heme sensitivity are unclear. We found that the accumulation of G-quadruplexes is affected by heme oxygenase-1 (Hmox1) expression, but in a cell-type-specific manner: hematopoietic stem cells (HSCs) from Hmox1−/− mice have upregulated expressions of G4-unwinding helicases (e.g., Brip1, Pif1) and show weaker staining for G-quadruplexes, whereas Hmox1-deficient murine induced pluripotent stem cells (iPSCs), despite the upregulation of helicases, have more G-quadruplexes, especially after exposure to exogenous heme. Using iPSCs expressing only nuclear or only cytoplasmic forms of Hmox1, we found that nuclear localization promotes G4 removal. We demonstrated that the proximity ligation assay (PLA) can detect cellular co-localization of G-quadruplexes with helicases, as well as with HMOX1, suggesting the potential role of HMOX1 in G4 modifications. However, this colocalization does not mean a direct interaction was detectable using the immunoprecipitation assay. Therefore, we concluded that HMOX1 influences G4 accumulation, but rather as one of the proteins regulating the heme availability, not as a rate-limiting factor. It is noteworthy that cellular G4–protein colocalizations can be quantitatively analyzed using PLA, even in rare cells.

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