scholarly journals Truncation of Tau selectively facilitates its pathological activities

2020 ◽  
Vol 295 (40) ◽  
pp. 13812-13828
Author(s):  
Jianlan Gu ◽  
Wen Xu ◽  
Nana Jin ◽  
Longfei Li ◽  
Yan Zhou ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Hyperphosphorylated Tau ◽  
Tau Pathology ◽  
Site Specific ◽  
Pathological Characteristics ◽  
Heparin Sulfate ◽  
Induced Aggregation ◽  
First Time ◽  
Self Aggregation

Neurofibrillary tangles of abnormally hyperphosphorylated Tau are a hallmark of Alzheimer's disease (AD) and related tauopathies. Tau is truncated at multiple sites by various proteases in AD brain. Although many studies have reported the effect of truncation on the aggregation of Tau, these studies mostly employed highly artificial conditions, using heparin sulfate or arachidonic acid to induce aggregation. Here, we report for the first time the pathological activities of various truncations of Tau, including site-specific phosphorylation, self-aggregation, binding to hyperphosphorylated and oligomeric Tau isolated from AD brain tissue (AD O-Tau), and aggregation seeded by AD O-Tau. We found that deletion of the first 150 or 230 amino acids (aa) enhanced Tau's site-specific phosphorylation, self-aggregation, and binding to AD O-Tau and aggregation seeded by AD O-Tau, but deletion of the first 50 aa did not produce a significant effect. Deletion of the last 50 aa was found to modulate Tau's site-specific phosphorylation, promote its self-aggregation, and cause it to be captured by and aggregation seeded by AD O-Tau, whereas deletion of the last 20 aa had no such effects. Among the truncated Taus, Tau151–391 showed the highest pathological activities. AD O-Tau induced aggregation of Tau151–391in vitro and in cultured cells. These findings suggest that the first 150 aa and the last 50 aa protect Tau from pathological characteristics and that their deletions facilitate pathological activities. Thus, inhibition of Tau truncation may represent a potential therapeutic approach to suppress Tau pathology in AD and related tauopathies.

scholarly journals Alzheimer’s disease brain contains tau fractions with differential prion-like activities

2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Longfei Li ◽  
Ruirui Shi ◽  
Jianlan Gu ◽  
Yunn Chyn Tung ◽  
Yan Zhou ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Cultured Cells ◽  
Regional Distribution ◽  
Proteinase K ◽  
Hyperphosphorylated Tau ◽  
Terminal Portion ◽  
Tau Pathology ◽  
Heat Stable ◽  
Tau Aggregation

AbstractNeurofibrillary tangles (NFTs) made of abnormally hyperphosphorylated tau are a hallmark of Alzheimer’s disease (AD) and related tauopathies. Regional distribution of NFTs is associated with the progression of the disease and has been proposed to be a result of prion-like propagation of misfolded tau. Tau in AD brain is heterogenous and presents in various forms. In the present study, we prepared different tau fractions by sedimentation combined with sarkosyl solubility from AD brains and analyzed their biochemical and pathological properties. We found that tau in oligomeric fraction (O-tau), sarkosyl-insoluble fractions 1 and 2 (SI1-tau and SI2-tau) and monomeric heat-stable fraction (HS-tau) showed differences in truncation, hyperphosphorylation, and resistance to proteinase K. O-tau, SI1-tau, and SI2-tau, but not HS-tau, were hyperphosphorylated at multiple sites and contained SDS- and β-mercaptoethanol–resistant high molecular weight aggregates, which lacked the N-terminal portion of tau. O-tau and SI2-tau displayed more truncation and less hyperphosphorylation than SI1-tau. Resistance to proteinase K was increased from O-tau to SI1-tau to SI2-tau. O-tau and SI1-tau, but not SI2-tau or HS-tau, captured tau from cell lysates and seeded tau aggregation in cultured cells. Heat treatment could not kill the prion-like activity of O-tau to capture normal tau. Hippocampal injection of O-tau into 18-month-old FVB mice induced significant tau aggregation in both ipsilateral and contralateral hippocampi, but SI1-tau only induced tau pathology in the ipsilateral hippocampus, and SI2-tau and HS-tau failed to induce any detectable tau aggregation. These findings suggest that O-tau and SI1-tau have prion-like activities and may serve as seeds to recruit tau and template tau to aggregate, resulting in the propagation of tau pathology. Heterogeneity of tau pathology within AD brain results in different fractions with different biological and prion-like properties, which may pose a major challenge in targeting tau for development of effective therapeutic treatments.

A Direct Comparison of Glassy Carbon and PEDOT-PSS Electrodes for High Charge Injection and Low Impedance Neural Interfaces

2016 ◽  
Vol 102 ◽  
pp. 68-76 ◽  
Author(s):  
Maria Vomero ◽  
Elisa Castagnola ◽  
Emma Maggiolini ◽  
Francesca Ciarpella ◽  
Irene Rembado ◽  
...  
Keyword(s):  
Electrochemical Performance ◽  
Glassy Carbon ◽  
Cultured Cells ◽  
Electrochemical Impedance ◽  
Neural Interfaces ◽  
Somatosensory Stimulation ◽  
Brain Surface ◽  
First Time

For neural applications, materials able to interface with the brain without harming it while recording high-fidelity signals over long-term implants are still sought after. Glassy Carbon (GC) and Poly (3,4-ethylenedioxythiophene)-poly (styrenesulfonate) (PEDOT-PSS) have proved to be promising materials for neural interfaces as they show – compared to conventional metal electrodes - higher conductivity, better electrochemical stability, very good mechanical properties and therefore seem to be very promising for in vivo applications. We present here, for the first time, a direct comparison between GC and PEDOT-PSS microelectrodes in terms of biocompatibility, electrical and electrochemical properties as well as in vivo recording capabilities, using electrocorticography microelectrode arrays located on flexible polyimide substrate. The GC microelectrodes were fabricated using a traditional negative lithography processes followed by pyrolysis. PEDOT-PSS was selectively electrodeposited on the desired electrodes. Electrochemical performance of the two materials was evaluated through electrochemical impedance spectroscopy and cyclic voltammetry. Biocompatibility was assessed through in-vitro studies evaluating cultured cells viability. The in vivo performance of the GC and PEDOT-PSS electrodes was directly compared by simultaneously recording neuronal activity during somatosensory stimulation in Long-Evans rats. We found that both GC and PEDOT-PSS electrodes outperform metals in terms of electrochemical performance and allow to obtain excellent recordings of somatosensory evoked potentials from the rat brain surface. Furthermore, we found that both GC and PEDOT-PSS substrates are highly biocompatible, confirming that they are safe for neural interface applications.

Microarray to reveal LncRNA profile and the role of lncCUEDC1 in BCSCs.

2018 ◽  
Vol 36 (5_suppl) ◽  
pp. 3-3
Author(s):  
Fengchun Zhang ◽  
Ying chun Xu ◽  
Ning Yan
Keyword(s):  
Breast Cancer ◽  
Cultured Cells ◽  
Bioinformatics Analyses ◽  
Functional Roles ◽  
Functional Activities ◽  
Non Coding Rnas ◽  
Microarray Analyses ◽  
First Time

3 Background: Recently, a growing amount of reports have shown that long non-coding RNAs (lncRNAs) are involved in breast cancer development, progression and metastasis. However, the correlation between lncRNAs and breast cancer stem cells (BCSCs) has been poorly explored. Methods: We initially isolated BCSCs from mammosphere-cultured cells; then, microarray analyses were carried out to detect the lncRNA signature in BCSCs. In addition, bioinformatics analyses, including Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG), were consulted to explore the functional roles of lncRNAs on BCSCs. Results: A total of 142 aberrantly expressed lncRNAs in BCSCs were identified, and of those lncRNAs, 25 were downregulated and 117 were upregulated compared to non-BCSCs. In addition, some of these lncRNAs were randomly selected and verified by RT-PCR and sanger sequencing. Notably, bioinformatics data showed that the lncRNAs that were detected were largely associated with stemness-related signalling pathways. Additionally, an interacting network between lncRNAs and its mRNAs was constructed to further depict the lncRNA functional activities. Furthermore, we found that lncCUEDC1 negatively regulated phenotype and biological functions of BCSCs in vitro. Conclusions: Our work establishes the lncRNAs signature in BCSCs for the first time. These findings provide us with evidence to explore the functionalities of lncRNAs in BCSCs and indicate that lncCUED1 is a prospective target for BCSCs.

scholarly journals Hsp22 with an N-Terminal Domain Truncation Mediates a Reduction in Tau Protein Levels

2020 ◽  
Vol 21 (15) ◽  
pp. 5442
Author(s):  
Jack M. Webster ◽  
April L. Darling ◽  
Taylor A. Sanders ◽  
Danielle M. Blazier ◽  
Yamile Vidal-Aguiar ◽  
...  
Keyword(s):  
Tau Protein ◽  
Cultured Cells ◽  
Inhibitory Effect ◽  
High Ratio ◽  
Protein Levels ◽  
Terminal Domain ◽  
Domain Truncation ◽  
Shock Proteins ◽  
Induced Aggregation

Misfolding, aggregation and accumulation of proteins are toxic elements in the progression of a broad range of neurodegenerative diseases. Molecular chaperones enable a cellular defense by reducing or compartmentalizing these insults. Small heat shock proteins (sHsps) engage proteins early in the process of misfolding and can facilitate their proper folding or refolding, sequestration, or clearance. Here, we evaluate the effects of the sHsp Hsp22, as well as a pseudophosphorylated mutant and an N-terminal domain deletion (NTDΔ) variant on tau aggregation in vitro and tau accumulation and aggregation in cultured cells. Hsp22 wild-type (WT) protein had a significant inhibitory effect on heparin-induced aggregation in vitro and the pseudophosphorylated mutant Hsp22 demonstrated a similar effect. When co-expressed in a cell culture model with tau, these Hsp22 constructs significantly reduced soluble tau protein levels when transfected at a high ratio relative to tau. However, the Hsp22 NTDΔ protein drastically reduced the soluble protein expression levels of both tau WT and tau P301L/S320F even at lower transfection ratios, which resulted in a correlative reduction of the triton-insoluble tau P301L/S320F aggregates.

scholarly journals Genetic Incorporation of Two Mutually Orthogonal Bioorthogonal Amino Acids That Enable Efficient Protein Dual-Labeling in Cells

2021 ◽  
Author(s):  
Riley M Bednar ◽  
Subhashis Jana ◽  
Sahiti Kuppa ◽  
Rachel Franklin ◽  
Joeseph Beckman ◽  
...  
Keyword(s):  
Amino Acids ◽  
Protein Function ◽  
Protein A ◽  
Noncanonical Amino Acids ◽  
Site Specific ◽  
Levels Of Detail ◽  
High Yields ◽  
First Time

The ability to site-specifically modify proteins at multiple sites in vivo will enable the study of protein function in its native environment with unprecedented levels of detail. Here, we present a versatile two-step strategy to meet this goal involving site-specific encoding of two distinct noncanonical amino acids bearing bioorthogonal handles into proteins in vivo followed by mutually orthogonal labeling. This general approach, that we call dual encoding and labeling (DEAL), allowed us to efficiently encoded tetrazine- and azide-bearing amino acids into a protein and demonstrate for the first time that the bioorthogonal labeling reactions with strained alkene and alkyne labels can function simultaneously and intracellularly with high yields when site-specifically encoded in a single protein. Using our DEAL system, we were able to perform topologically-defined protein-protein crosslinking, intramolecular stapling, and site-specific installation of fluorophores all inside living Escherichia coli cells, as well as study the DNA-binding properties of yeast Replication Protein A in vitro. By enabling the efficient dual modification of proteins in vivo, this DEAL approach provides a tool for the characterization and engineering of proteins in vivo.

scholarly journals Selective Cleavage of AAVS1 Substrates by the Adeno-Associated Virus Type 2 Rep68 Protein Is Dependent on Topological and Sequence Constraints

2000 ◽  
Vol 74 (19) ◽  
pp. 8831-8842 ◽  
Author(s):  
Stefania Lamartina ◽  
Gennaro Ciliberto ◽  
Carlo Toniatti
Keyword(s):  
Virus Type ◽  
Adeno Associated Virus ◽  
Site Specific ◽  
Site Specific Integration ◽  
A Site ◽  
First Time ◽  
Sequence Constraints

ABSTRACT The adeno-associated virus type 2 (AAV-2) Rep78 and Rep68 proteins are required for replication of the virus as well as its site-specific integration into a unique site, called AAVS1, of human chromosome 19. Rep78 and Rep68 initiate replication by binding to a Rep binding site (RBS) contained in the AAV-2 inverted terminal repeats (ITRs) and then specifically nicking at a nearby site called the terminal resolution site (trs). Similarly, Rep78 and Rep68 are postulated to trigger the integration process by binding and nicking RBS andtrs homologues present in AAVS1. However, Rep78 and Rep68 cleave in vitro AAVS1 duplex-linear substrates much less efficiently than hairpinned ITRs. In this study, we show that the AAV-2 Rep68 endonuclease activity is affected by the topology of the substrates in that it efficiently cleaves in vitro in a site- and strand-specific manner the AAVS1 trs only if this sequence is in a supercoiled (SC) conformation. DNA sequence mutagenesis in the context of SC templates allowed us to elucidate for the first time the AAVS1trs sequence and position requirements for Rep68-mediated cleavage. Interestingly, Rep68 did not cleave SC templates containing RBS from other sites of the human genome. These findings have intriguing implications for AAV-2 site-specific integration in vivo.

scholarly journals DDX17 promotes hepatocellular carcinoma progression via inhibiting Klf4 transcriptional activity

2019 ◽  
Vol 10 (11) ◽  
Author(s):  
Ying Xue ◽  
Xuebing Jia ◽  
Changcan Li ◽  
Ke Zhang ◽  
Lei Li ◽  
...  
Keyword(s):  
Transcriptional Activity ◽  
Target Gene ◽  
Target Genes ◽  
The Cancer Genome Atlas ◽  
Gene Expressions ◽  
Pathological Characteristics ◽  
Dead Box ◽  
Cancer Genome Atlas ◽  
First Time

Abstract DEAD box RNA helicase 17 (DDX17) is a transcriptional regulator of several transcription factors, which is more appreciated than its role in RNA metabolism. However, prognostic value and biofunction of DDX17 in HCC remain unclear. Illuminating the mechanism underlying the regulating HCC progression by DDX17 may contribute to therapeutic strategies. In our study, we report for the first time that DDX17 was overexpressed in HCC specimens by using The Cancer Genome Atlas (TCGA) and immunohistochemistry (IHC) and correlated to clinical pathological characteristics and patients’ survival. In vitro, DDX17 was ascertained to alter HCC migratory and invasive capacities after overexpression and knockdown in HCC cell lines. Moreover, by performing co-immunoprecipitation (Co-IP) and GST-pull down assay, the physical association between DDX17 and Klf4 was discovered and validated. Additionally, DDX17 could modulate expressions of Klf4 target genes including E-cadherin, MMP2 by inhibiting the promoter activity. The potent correlation between DDX17 and Klf4 target gene expressions was further appraised by a same set of 30 HCC tissues. Besides, we discovered that DDX17 could not deploy its function in regulating Klf4 target gene expressions and HCC progression in Klf4-depletion condition. Intriguingly, DDX17 failed to interact with Klf4 once the zinc-finger domain was deleted and inhibited the binding of Klf4 on MMP-2 promoter. Collectively, our study enucleates novel mechanism of DDX17-mediated oncogenesis by suppressing the transcriptional activity of Klf4 thus is likely to be a therapeutic target in HCC.

Ultrastructural Correlations between Clonal Human Tumor Colonies and in Vivo Neoplasms

1982 ◽  
Vol 40 ◽  
pp. 210-211
Author(s):  
M.J. Murphy ◽  
R.R. Price ◽  
J.C. Sloman
Keyword(s):  
Cultured Cells ◽  
Human Tumor ◽  
Cell Type ◽  
Tumor Mass ◽  
Initial Cell ◽  
Cloning Assay ◽  
Human Tumor Cloning ◽  
The Relationship

The in vitro human tumor cloning assay originally described by Salmon and Hamburger has been applied recently to the investigation of differential anti-tumor drug sensitivities over a broad range of human neoplasms. A major problem in the acceptance of this technique has been the question of the relationship between the cultured cells and the original patient tumor, i.e., whether the colonies that develop derive from the neoplasm or from some other cell type within the initial cell population. A study of the ultrastructural morphology of the cultured cells vs. patient tumor has therefore been undertaken to resolve this question. Direct correlation was assured by division of a common tumor mass at surgical resection, one biopsy being fixed for TEM studies, the second being rapidly transported to the laboratory for culture.

Preparation of cultured astrocytes for x-ray microanalysis

1989 ◽  
Vol 47 ◽  
pp. 988-989
Author(s):  
N.K.R. Smith ◽  
K.E. Hunter ◽  
P. Mobley ◽  
L.P. Felpel
Keyword(s):  
Cultured Cells ◽  
Elemental Content ◽  
Elemental Distribution ◽  
Sprague Dawley ◽  
X Ray ◽  
Cultured Astrocytes ◽  
Freeze Dried ◽  
Ultimate Objective

Electron probe energy dispersive x-ray microanalysis (XRMA) offers a powerful tool for the determination of intracellular elemental content of biological tissue. However, preparation of the tissue specimen , particularly excitable central nervous system (CNS) tissue , for XRMA is rather difficult, as dissection of a sample from the intact organism frequently results in artefacts in elemental distribution. To circumvent the problems inherent in the in vivo preparation, we turned to an in vitro preparation of astrocytes grown in tissue culture. However, preparations of in vitro samples offer a new and unique set of problems. Generally, cultured cells, growing in monolayer, must be harvested by either mechanical or enzymatic procedures, resulting in variable degrees of damage to the cells and compromised intracel1ular elemental distribution. The ultimate objective is to process and analyze unperturbed cells. With the objective of sparing others from some of the same efforts, we are reporting the considerable difficulties we have encountered in attempting to prepare astrocytes for XRMA.Tissue cultures of astrocytes from newborn C57 mice or Sprague Dawley rats were prepared and cultured by standard techniques, usually in T25 flasks, except as noted differently on Cytodex beads or on gelatin. After different preparative procedures, all samples were frozen on brass pins in liquid propane, stored in liquid nitrogen, cryosectioned (0.1 μm), freeze dried, and microanalyzed as previously reported.

Ultrastructural investigations of MDL 19,660-induced effects on the canine platelet and megakaryocyte

1990 ◽  
Vol 48 (3) ◽  
pp. 374-375
Author(s):  
D.E. Loudy ◽  
J. Sprinkle-Cavallo ◽  
J.T. Yarrington ◽  
F.Y. Thompson ◽  
J.P. Gibson
Keyword(s):  
Antidepressant Drug ◽  
Beagle Dogs ◽  
Long Term Effects ◽  
Short Term ◽  
Platelet Counts ◽  
Toxicological Studies ◽  
Induced Aggregation ◽  
Internal Architecture

Previous short term toxicological studies of one to two weeks duration have demonstrated that MDL 19,660 (5-(4-chlorophenyl)-2,4-dihydro-2,4-dimethyl-3Hl, 2,4-triazole-3-thione), an antidepressant drug, causes a dose-related thrombocytopenia in dogs. Platelet counts started to decline after two days of dosing with 30 mg/kg/day and continued to decrease to their lowest levels by 5-7 days. The loss in platelets was primarily of the small discoid subpopulation. In vitro studies have also indicated that MDL 19,660: does not spontaneously aggregate canine platelets and has moderate antiaggregating properties by inhibiting ADP-induced aggregation. The objectives of the present investigation of MDL 19,660 were to evaluate ultrastructurally long term effects on platelet internal architecture and changes in subpopulations of platelets and megakaryocytes.Nine male and nine female beagle dogs were divided equally into three groups and were administered orally 0, 15, or 30 mg/kg/day of MDL 19,660 for three months. Compared to a control platelet range of 353,000- 452,000/μl, a doserelated thrombocytopenia reached a maximum severity of an average of 135,000/μl for the 15 mg/kg/day dogs after two weeks and 81,000/μl for the 30 mg/kg/day dogs after one week.

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