scholarly journals ­TREM2-H157Y Increases Soluble TREM2 Production and Reduces Amyloid Pathology

2021 ◽  
Author(s):  
Wenhui Qiao ◽  
Yixing Chen ◽  
Yuka A. Martens ◽  
Chia-Chen Liu ◽  
Joshua A. Knight ◽  
...  
Keyword(s):  
Ectopic Expression ◽  
Proteolytic Processing ◽  
Synaptic Function ◽  
Hek293 Cells ◽  
Immunofluorescent Staining ◽  
Amyloid Pathology ◽  
Biochemical Assays ◽  
Soluble Trem2

Abstract Background: The p.H157Y variant of TREM2 (Triggering Receptor Expressed on Myeloid Cells 2) has been reported to increase Alzheimer’s disease (AD) risk. This mutation in the extracellular domain of TREM2 localizes at the cleavage site and was shown to enhance shedding and impair phagocytosis in vitro by ectopic expression of TREM2-H157Y in HEK293 cells. However, the physiological and AD-related outcomes of TREM2 H157Y mutation in vivo remain unknown.Methods: We generated a novel Trem2 H157Y knock-in mouse model through CRISPR-Cas9 technology and investigated how Trem2 H157Y mutation impacts TREM2 proteolytic processing, synaptic function, and AD-related amyloid pathology by conducting biochemical assays, immunofluorescent staining, hippocampal electrophysiology, and in vivo micro-dialysis in awake, free-moving animals.Results: Consistent with previous in vitro findings, TREM2-H157Y increases the amount of soluble TREM2 (sTREM2) in the cortex and serum of mutant mice compared to the wild type controls. Interestingly, the Trem2 H157Y variant enhances synaptic plasticity without affecting microglial density and morphology. In the presence of amyloid pathology, TREM2-H157Y surprisingly accelerates Aβ clearance and reduces amyloid burden and microgliosis. Conclusion: Taken together, our findings support a beneficial effect of the Trem2 H157Y mutation in synaptic function and in mitigating amyloid pathology. Considering the genetic association of TREM2 p.H157Y with AD, we speculate TREM2-H157Y might increase AD risk through an amyloid-independent pathway, as such its effects on tauopathy and neurodegeneration merit further investigation.

scholarly journals TREM2-H157Y Increases Soluble TREM2 Production and Reduces Amyloid Pathology

2021 ◽  
Author(s):  
Wenhui Qiao ◽  
Yixing Chen ◽  
Yuka Martens ◽  
Chia-chen Liu ◽  
Joshua Knight ◽  
...  
Keyword(s):  
Synaptic Plasticity ◽  
Mouse Model ◽  
Proteolytic Processing ◽  
Synaptic Function ◽  
Amyloid Burden ◽  
Wild Type ◽  
Amyloid Pathology ◽  
Soluble Trem2 ◽  
Aβ Clearance

The p.H157Y variant of TREM2 (Triggering Receptor Expressed on Myeloid Cells 2) has been reported to increase Alzheimer's disease (AD) risk. This mutation in the extracellular domain of TREM2 localizes at the cleavage site, leading to enhanced shedding. Here, we generated a novel Trem2 H157Y knock-in mouse model to investigate how this H157Y mutation impacts TREM2 proteolytic processing, synaptic function, and AD-related amyloid pathology. Consistent with previous in vitro findings, TREM2-H157Y increases the amount of soluble TREM2 (sTREM2) in the cortex and serum of mutant mice compared to the wild type controls. Interestingly, the Trem2 H157Y variant enhances synaptic plasticity without affecting microglial density and morphology. In the presence of amyloid pathology, TREM2-H157Y surprisingly accelerates Aβ clearance and reduces amyloid burden and microgliosis. Taken together, our findings support a beneficial effect of the Trem2 H157Y mutation in synaptic function and in mitigating amyloid pathology. Considering the genetic association of TREM2 p.H157Y with AD, we speculate TREM2-H157Y might increase AD risk through an amyloid-independent pathway, as such its effects on tauopathy and neurodegeneration merit further investigation.

Mutants of cubitus interruptus that are independent of PKA regulation are independent of hedgehog signaling

Development ◽  
1999 ◽  
Vol 126 (16) ◽  
pp. 3607-3616 ◽  
Author(s):  
Y. Chen ◽  
J.R. Cardinaux ◽  
R.H. Goodman ◽  
S.M. Smolik
Keyword(s):  
Gene Expression ◽  
Target Gene ◽  
Ectopic Expression ◽  
Proteolytic Processing ◽  
Dominant Negative ◽  
Cubitus Interruptus ◽  
Target Gene Expression ◽  
Exogenous Expression

Hedgehog (HH) is an important morphogen involved in pattern formation during Drosophila embryogenesis and disc development. cubitus interruptus (ci) encodes a transcription factor responsible for transducing the hh signal in the nucleus and activating hh target gene expression. Previous studies have shown that CI exists in two forms: a 75 kDa proteolytic repressor form and a 155 kDa activator form. The ratio of these forms, which is regulated positively by hh signaling and negatively by PKA activity, determines the on/off status of hh target gene expression. In this paper, we demonstrate that the exogenous expression of CI that is mutant for four consensus PKA sites [CI(m1-4)], causes ectopic expression of wingless (wg) in vivo and a phenotype consistent with wg overexpression. Expression of CI(m1-4), but not CI(wt), can rescue the hh mutant phenotype and restore wg expression in hh mutant embryos. When PKA activity is suppressed by expressing a dominant negative PKA mutant, the exogenous expression of CI(wt) results in overexpression of wg and lethality in embryogenesis, defects that are similar to those caused by the exogenous expression of CI(m1-4). In addition, we demonstrate that, in cell culture, the mutation of any one of the three serine-containing PKA sites abolishes the proteolytic processing of CI. We also show that PKA directly phosphorylates the four consensus phosphorylation sites in vitro. Taken together, our results suggest that positive hh and negative PKA regulation of wg gene expression converge on the regulation of CI phosphorylation.

scholarly journals Calcium Binding of ARC Mediates Regulation of Caspase 8 and Cell Death

2004 ◽  
Vol 24 (22) ◽  
pp. 9763-9770 ◽  
Author(s):  
Dong-Gyu Jo ◽  
Joon-Il Jun ◽  
Jae-Woong Chang ◽  
Yeon-Mi Hong ◽  
Sungmin Song ◽  
...  
Keyword(s):  
Cell Death ◽  
Calcium Binding ◽  
Ectopic Expression ◽  
Hek293 Cells ◽  
Caspase 8 ◽  
Cytotoxic Effects ◽  
Protein Protein Interaction ◽  
Rich Domain

ABSTRACT Apoptosis repressor with CARD (ARC) possesses the ability not only to block activation of caspase 8 but to modulate caspase-independent mitochondrial events associated with cell death. However, it is not known how ARC modulates both caspase-dependent and caspase-independent cell death. Here, we report that ARC is a Ca2+-dependent regulator of caspase 8 and cell death. We found that in Ca2+ overlay and Stains-all assays, ARC protein bound to Ca2+ through the C-terminal proline/glutamate-rich (P/E-rich) domain. ARC expression reduced not only cytosolic Ca2+ transients but also cytotoxic effects of thapsigargin, A23187, and ionomycin, for which the Ca2+-binding domain of ARC was indispensable. Conversely, direct interference of endogenous ARC synthesis by targeting ARC enhanced such Ca2+-mediated cell death. In addition, binding and immunoprecipitation analyses revealed that the protein-protein interaction between ARC and caspase 8 was decreased by the increase of Ca2+ concentration in vitro and by the treatment of HEK293 cells with thapsigargin in vivo. Caspase 8 activation was also required for the thapsigargin-induced cell death and suppressed by the ectopic expression of ARC. These results suggest that calcium binding mediates regulation of caspase 8 and cell death by ARC.

ACIS, A Novel KepTide™, Binds to ACE-2 Receptor and Inhibits the Infection of SARS-CoV2 Virus in vitro in Primate Kidney Cells: Therapeutic Implications for COVID-19 (Preprint)

2020 ◽  
Author(s):  
Avik Sotira Scientific
Keyword(s):  
Social Life ◽  
Plaque Assay ◽  
Host Cells ◽  
Surface Glycoprotein ◽  
Crystallographic Structure ◽  
Therapeutic Implications ◽  
Biochemical Assays ◽  
Targeted Medicine

UNSTRUCTURED Coronavirus disease 2019 (COVID-19) is a severe acute respiratory syndrome (SARS) caused by a virus known as SARS-Coronavirus 2 (SARS-CoV2). Without a targeted-medicine, this disease has been causing a massive humanitarian crisis not only in terms of mortality, but also imposing a lasting damage to social life and economic progress of humankind. Therefore, an immediate therapeutic strategy needs to be intervened to mitigate this global crisis. Here, we report a novel KepTide™ (Knock-End Peptide) therapy that nullifies SARS-CoV2 infection. SARS-CoV2 employs its surface glycoprotein “spike” (S-glycoprotein) to interact with angiotensin converting enzyme-2 (ACE-2) receptor for its infection in host cells. Based on our in-silico-based homology modeling study validated with a recent X-ray crystallographic structure (PDB ID:6M0J), we have identified that a conserved motif of S-glycoprotein that intimately engages multiple hydrogen-bond (H-bond) interactions with ACE-2 enzyme. Accordingly, we designed a peptide, termed as ACIS (ACE-2 Inhibitory motif of Spike), that displayed significant affinity towards ACE-2 enzyme as confirmed by biochemical assays such as BLItz and fluorescence polarization assays. Interestingly, more than one biochemical modifications were adopted in ACIS in order to enhance the inhibitory action of ACIS and hence called as KEpTide™. Consequently, a monolayer invasion assay, plaque assay and dual immunofluorescence analysis further revealed that KEpTide™ efficiently mitigated the infection of SARS-CoV2 in vitro in VERO E6 cells. Finally, evaluating the relative abundance of ACIS in lungs and the potential side-effects in vivo in mice, our current study discovers a novel KepTide™ therapy that is safe, stable, and robust to attenuate the infection of SARS-CoV2 virus if administered intranasally. INTERNATIONAL REGISTERED REPORT RR2-https://doi.org/10.1101/2020.10.13.337584

scholarly journals Assessment of SnFe2O4 Nanoparticles for Potential Application in Theranostics: Synthesis, Characterization, In Vitro, and In Vivo Toxicity

Materials ◽  
2021 ◽  
Vol 14 (4) ◽  
pp. 825
Author(s):  
Saman Sargazi ◽  
Mohammad Reza Hajinezhad ◽  
Abbas Rahdar ◽  
Muhammad Nadeem Zafar ◽  
Aneesa Awan ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
A549 Cells ◽  
In Vitro Cytotoxicity ◽  
Hek293 Cells ◽  
Hydrothermal Route ◽  
X Ray ◽  
Tin Chloride ◽  
Bet Analysis

In this research, tin ferrite (SnFe2O4) NPs were synthesized via hydrothermal route using ferric chloride and tin chloride as precursors and were then characterized in terms of morphology and structure using Fourier-transform infrared spectroscopy (FTIR), Ultraviolet–visible spectroscopy (UV-Vis), X-ray power diffraction (XRD), Scanning electron microscopy (SEM), Transmission electron microscopy (TEM), and Brunauer–Emmett–Teller (BET) method. The obtained UV-Vis spectra was used to measure band gap energy of as-prepared SnFe2O4 NPs. XRD confirmed the spinel structure of NPs, while SEM and TEM analyses disclosed the size of NPs in the range of 15–50 nm and revealed the spherical shape of NPs. Moreover, energy dispersive X-ray spectroscopy (EDS) and BET analysis was carried out to estimate elemental composition and specific surface area, respectively. In vitro cytotoxicity of the synthesized NPs were studied on normal (HUVEC, HEK293) and cancerous (A549) human cell lines. HUVEC cells were resistant to SnFe2O4 NPs; while a significant decrease in the viability of HEK293 cells was observed when treated with higher concentrations of SnFe2O4 NPs. Furthermore, SnFe2O4 NPs induced dramatic cytotoxicity against A549 cells. For in vivo study, rats received SnFe2O4 NPs at dosages of 0, 0.1, 1, and 10 mg/kg. The 10 mg/kg dose increased serum blood urea nitrogen and creatinine compared to the controls (P < 0.05). The pathology showed necrosis in the liver, heart, and lungs, and the greatest damages were related to the kidneys. Overall, the in vivo and in vitro experiments showed that SnFe2O4 NPs at high doses had toxic effects on lung, liver and kidney cells without inducing toxicity to HUVECs. Further studies are warranted to fully elucidate the side effects of SnFe2O4 NPs for their application in theranostics.

scholarly journals Human Keratinocytes Adopt Neuronal Fates After In Utero Transplantation in the Developing Rat Brain

2021 ◽  
Vol 30 ◽  
pp. 096368972097821
Author(s):  
Andrea Tenorio-Mina ◽  
Daniel Cortés ◽  
Joel Esquivel-Estudillo ◽  
Adolfo López-Ornelas ◽  
Alejandro Cabrera-Wrooman ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Ectopic Expression ◽  
Neural Induction ◽  
Human Keratinocytes ◽  
Differentiation Potential ◽  
Neuronal Proteins ◽  
Green Fluorescent

Human skin contains keratinocytes in the epidermis. Such cells share their ectodermal origin with the central nervous system (CNS). Recent studies have demonstrated that terminally differentiated somatic cells can adopt a pluripotent state, or can directly convert its phenotype to neurons, after ectopic expression of transcription factors. In this article we tested the hypothesis that human keratinocytes can adopt neural fates after culturing them in suspension with a neural medium. Initially, keratinocytes expressed Keratins and Vimentin. After neural induction, transcriptional upregulation of NESTIN, SOX2, VIMENTIN, SOX1, and MUSASHI1 was observed, concomitant with significant increases in NESTIN detected by immunostaining. However, in vitro differentiation did not yield the expression of neuronal or astrocytic markers. We tested the differentiation potential of control and neural-induced keratinocytes by grafting them in the developing CNS of rats, through ultrasound-guided injection. For this purpose, keratinocytes were transduced with lentivirus that contained the coding sequence of green fluorescent protein. Cell sorting was employed to select cells with high fluorescence. Unexpectedly, 4 days after grafting these cells in the ventricles, both control and neural-induced cells expressed green fluorescent protein together with the neuronal proteins βIII-Tubulin and Microtubule-Associated Protein 2. These results support the notion that in vivo environment provides appropriate signals to evaluate the neuronal differentiation potential of keratinocytes or other non-neural cell populations.

scholarly journals Two Drosophila Innexins Are Expressed in Overlapping Domains and Cooperate to Form Gap-Junction Channels

2000 ◽  
Vol 11 (7) ◽  
pp. 2459-2470 ◽  
Author(s):  
Lucy A. Stebbings ◽  
Martin G. Todman ◽  
Pauline Phelan ◽  
Jonathan P. Bacon ◽  
Jane A. Davies
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Ectopic Expression ◽  
In Vivo Studies ◽  
Electrophysiological Properties ◽  
Gap Junction Channels ◽  
Overlapping Domains ◽  
In Vitro Data

Members of the innexin protein family are structural components of invertebrate gap junctions and are analogous to vertebrate connexins. Here we investigate two Drosophila innexin genes,Dm-inx2 and Dm-inx3 and show that they are expressed in overlapping domains throughout embryogenesis, most notably in epidermal cells bordering each segment. We also explore the gap-junction–forming capabilities of the encoded proteins. In pairedXenopus oocytes, the injection of Dm-inx2mRNA results in the formation of voltage-sensitive channels in only ∼ 40% of cell pairs. In contrast, Dm-Inx3 never forms channels. Crucially, when both mRNAs are coexpressed, functional channels are formed reliably, and the electrophysiological properties of these channels distinguish them from those formed by Dm-Inx2 alone. We relate these in vitro data to in vivo studies. Ectopic expression ofDm-inx2 in vivo has limited effects on the viability ofDrosophila, and animals ectopically expressingDm-inx3 are unaffected. However, ectopic expression of both transcripts together severely reduces viability, presumably because of the formation of inappropriate gap junctions. We conclude that Dm-Inx2 and Dm-Inx3, which are expressed in overlapping domains during embryogenesis, can form oligomeric gap-junction channels.

scholarly journals The ectopic expression of Arabidopsis glucosyltransferase UGT74D1 affects leaf positioning through modulating indole-3-acetic acid homeostasis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Shanghui Jin ◽  
Bingkai Hou ◽  
Guizhi Zhang
Keyword(s):  
Acetic Acid ◽  
Ectopic Expression ◽  
Leaf Angle ◽  
Kinase Substrate ◽  
Auxin Distribution ◽  
Leaf Tissues ◽  
Photosynthesis Efficiency ◽  
Indole 3 Acetic Acid

AbstractLeaf angle is an important agronomic trait affecting photosynthesis efficiency and crop yield. Although the mechanisms involved in the leaf angle control are intensively studied in monocots, factors contribute to the leaf angle in dicots are largely unknown. In this article, we explored the physiological roles of an Arabidopsis glucosyltransferase, UGT74D1, which have been proved to be indole-3-acetic acid (IAA) glucosyltransferase in vitro. We found that UGT74D1 possessed the enzymatic activity toward IAA glucosylation in vivo and its expression was induced by auxins. The ectopically expressed UGT74D1 obviously reduced the leaf angle with an altered IAA level, auxin distribution and cell size in leaf tissues. The expression of several key genes involved in the leaf shaping and leaf positioning, including PHYTOCHROME KINASE SUBSTRATE (PKS) genes and TEOSINTE BRANCHED1, CYCLOIDEA, and PCF (TCP) genes, were dramatically changed by ectopic expression of UGT74D1. In addition, clear transcription changes of YUCCA genes and other auxin related genes can be observed in overexpression lines. Taken together, our data indicate that glucosyltransferase UGT74D1 could affect leaf positioning through modulating auxin homeostasis and regulating transcription of PKS and TCP genes, suggesting a potential new role of UGT74D1 in regulation of leaf angle in dicot Arabidopsis.

scholarly journals The Protective Effect of Sika Deer Antler Protein on Gentamicin-Induced Nephrotoxicity in Vitro and in Vivo

2018 ◽  
Vol 50 (3) ◽  
pp. 841-850 ◽  
Author(s):  
Hang Sun ◽  
Huihai Yang ◽  
Haonan Ruan ◽  
Wei Li ◽  
Xinhong He ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Sika Deer ◽  
Kidney Injury ◽  
Hek293 Cells ◽  
Inflammatory Factors ◽  
Oxidative Stress Marker ◽  
Renal Protection ◽  
Deer Antler

Background/Aims: Sika deer (Cervus nippon Temminck) antler is traditional animal medicine of renal protection in East Asia. This study measured the effect of sika deer antler protein (SDAPR) on gentamicin (GM)-induced cytotoxicity in HEK293 cells, and investigated the effect of SDAPR against GM-induced nephrotoxicity in mice. Methods: HEK293 cells viability and oxidative stress were measured in HEK293 cells while flow cytometry was used for apoptosis analysis. The acute kidney injury biomarkers, kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL) and cystatin c (Cys-C), were repeatedly measured by ELISA assay. ICR male mice were randomly assigned six groups: Control, GM with vehicle, single SDAPR, GM with SDAPR at three concentrations 50, 100, 200 mg/kg/d, p.o., 10 d. GM was injected for 8 consecutive days (100 mg/kg/d, i.p.). Renal function, oxidative stress and levels of inflammatory factors were measured in vivo. Renal tissues were stained with H&E to observe pathological changes. Results: Pretreatment with SDAPR (0.5-4.0 mg/mL) significantly improved cell viability. Treatment with SDAPR could reduce KIM-1, NGAL and Cys-C activity. SDAPR could improve antioxidant defense and attenuated apoptosis on HEK293 cells. SDAPR also ameliorated GM-induced histopathologic changes, and decreased blood urea nitrogen (BUN) and serum creatinine (Cr). Additionally, SDAPR significantly regulated oxidative stress marker and interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) inflammatory cytokines. Conclusion: These results show that SDAPR could be an effective dietary supplement to relieve GM-induced nephrotoxicity by improved antioxidase activity, suppressed inflammation, and inhibited apoptosis in vitro and vivo.

scholarly journals Hypermethylation of DHRS3 as a Novel Tumor Suppressor Involved in Tumor Growth and Prognosis in Gastric Cancer

2021 ◽  
Vol 9 ◽  
Author(s):  
Sha Sumei ◽  
Kong Xiangyun ◽  
Chen Fenrong ◽  
Sun Xueguang ◽  
Hu Sijun ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Tumor Suppressor ◽  
Tumor Growth ◽  
Human Cancer ◽  
Methylation Status ◽  
Ectopic Expression ◽  
Survival Times

Background/AimsThe role of DHRS3 in human cancer remains unclear. Our study explored the role of DHRS3 in gastric cancer (GC) and its clinicopathological significance and associated mechanisms.MaterialsBisulfite-assisted genomic sequencing PCR and a Mass-Array system were used to evaluate and quantify the methylation levels of the promoter. The expression levels and biological function of DHRS3 was examined by both in vitro and in vivo assays. A two-way hierarchical cluster analysis was used to classify the methylation profiles, and the correlation between the methylation status of the DHRS3 promoter and the clinicopathological characteristics of GC were then assessed.ResultsThe DHRS3 promoter was hypermethylated in GC samples, while the mRNA and protein levels of DHRS3 were significantly downregulated. Ectopic expression of DHRS3 in GC cells inhibited cell proliferation and migration in vitro, decreased tumor growth in vivo. DHRS3 methylation was correlated with histological type and poor differentiation of tumors. GC patients with high degrees of CpG 9.10 methylation had shorter survival times than those with lower methylation.ConclusionDHRS3 was hypermethylated and downregulated in GC patients. Reduced expression of DHRS3 is implicated in gastric carcinogenesis, which suggests DHRS3 is a tumor suppressor.

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