scholarly journals DJ-1 inhibits microglial activation and protects dopaminergic neurons in vitro and in vivo through interacting with microglial p65

2021 ◽  
Vol 12 (8) ◽  
Author(s):  
Zixuan Lin ◽  
Chen Chen ◽  
Dongqin Yang ◽  
Jianqing Ding ◽  
Guanghui Wang ◽  
...  
Keyword(s):  
Microglial Activation ◽  
Nuclear Translocation ◽  
Substantia Nigra Pars Compacta ◽  
Loss Of Function ◽  
Wild Type ◽  
Wild Type Littermate ◽  
Cellular Models ◽  
Bv2 Cells

AbstractParkinson’s disease (PD), one of the most common neurodegenerative disorders, is characterized by progressive neurodegeneration of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc). DJ-1 acts essential roles in neuronal protection and anti-neuroinflammatory response, and its loss of function is tightly associated with a familial recessive form of PD. However, the molecular mechanism of DJ-1 involved in neuroinflammation is largely unclear. Here, we found that wild-type DJ-1, rather than the pathogenic L166P mutant DJ-1, directly binds to the subunit p65 of nuclear factor-κB (NF-κB) in the cytoplasm, and loss of DJ-1 promotes p65 nuclear translocation by facilitating the dissociation between p65 and NF-κB inhibitor α (IκBα). DJ-1 knockout (DJ-1−/−) mice exhibit more microglial activation compared with wild-type littermate controls, especially in response to lipopolysaccharide (LPS) treatment. In cellular models, knockdown of DJ-1 significantly upregulates the gene expression and increases the release of LPS-treated inflammatory cytokines in primary microglia and BV2 cells. Furthermore, DJ-1 deficiency in microglia significantly enhances the neuronal toxicity in response to LPS stimulus. In addition, pharmacological blockage of NF-κB nuclear translocation by SN-50 prevents microglial activation and alleviates the damage of DA neurons induced by microglial DJ-1 deficiency in vivo and in vitro. Thus, our data illustrate a novel mechanism by which DJ-1 facilitates the interaction between IκBα and p65 by binding to p65 in microglia, and thus repressing microglial activation and exhibiting the protection of DA neurons from neuroinflammation-mediated injury in PD.

scholarly journals DJ-1 regulates the integrity and function of ER-mitochondria association through interaction with IP3R3-Grp75-VDAC1

2019 ◽  
Vol 116 (50) ◽  
pp. 25322-25328 ◽  
Author(s):  
Yi Liu ◽  
Xiaopin Ma ◽  
Hisashi Fujioka ◽  
Jun Liu ◽  
Shengdi Chen ◽  
...  
Keyword(s):  
Autosomal Recessive ◽  
Cellular Mechanism ◽  
Loss Of Function ◽  
Wild Type ◽  
Calcium Efflux ◽  
And Function ◽  
The Brain ◽  
Early Onset Parkinson’S Disease

Loss-of-function mutations in DJ-1 are associated with autosomal recessive early onset Parkinson’s disease (PD), yet the underlying pathogenic mechanism remains elusive. Here we demonstrate that DJ-1 localized to the mitochondria-associated membrane (MAM) both in vitro and in vivo. In fact, DJ-1 physically interacts with and is an essential component of the IP3R3-Grp75-VDAC1 complexes at MAM. Loss of DJ-1 disrupted the IP3R3-Grp75-VDAC1 complex and led to reduced endoplasmic reticulum (ER)-mitochondria association and disturbed function of MAM and mitochondria in vitro. These deficits could be rescued by wild-type DJ-1 but not by the familial PD-associated L166P mutant which had demonstrated reduced interaction with IP3R3-Grp75. Furthermore, DJ-1 ablation disturbed calcium efflux-induced IP3R3 degradation after carbachol treatment and caused IP3R3 accumulation at the MAM in vitro. Importantly, similar deficits in IP3R3-Grp75-VDAC1 complexes and MAM were found in the brain of DJ-1 knockout mice in vivo. The DJ-1 level was reduced in the substantia nigra of sporadic PD patients, which was associated with reduced IP3R3-DJ-1 interaction and ER-mitochondria association. Together, these findings offer insights into the cellular mechanism in the involvement of DJ-1 in the regulation of the integrity and calcium cross-talk between ER and mitochondria and suggests that impaired ER-mitochondria association could contribute to the pathogenesis of PD.

scholarly journals Regulation of cell proliferation by ERK and signal-dependent nuclear translocation of ERK is dependent on Tm5NM1-containing actin filaments

2015 ◽  
Vol 26 (13) ◽  
pp. 2475-2490 ◽  
Author(s):  
Galina Schevzov ◽  
Anthony J. Kee ◽  
Bin Wang ◽  
Vanessa B. Sequeira ◽  
Jeff Hook ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Actin Filaments ◽  
Nuclear Translocation ◽  
Genetic Manipulation ◽  
Wild Type ◽  
Proximity Ligation ◽  
Mouse Embryo Fibroblasts ◽  
Wild Type Mefs

ERK-regulated cell proliferation requires multiple phosphorylation events catalyzed first by MEK and then by casein kinase 2 (CK2), followed by interaction with importin7 and subsequent nuclear translocation of pERK. We report that genetic manipulation of a core component of the actin filaments of cancer cells, the tropomyosin Tm5NM1, regulates the proliferation of normal cells both in vitro and in vivo. Mouse embryo fibroblasts (MEFs) lacking Tm5NM1, which have reduced proliferative capacity, are insensitive to inhibition of ERK by peptide and small-molecule inhibitors, indicating that ERK is unable to regulate proliferation of these knockout (KO) cells. Treatment of wild-type MEFs with a CK2 inhibitor to block phosphorylation of the nuclear translocation signal in pERK resulted in greatly decreased cell proliferation and a significant reduction in the nuclear translocation of pERK. In contrast, Tm5NM1 KO MEFs, which show reduced nuclear translocation of pERK, were unaffected by inhibition of CK2. This suggested that it is nuclear translocation of CK2-phosphorylated pERK that regulates cell proliferation and this capacity is absent in Tm5NM1 KO cells. Proximity ligation assays confirmed a growth factor–stimulated interaction of pERK with Tm5NM1 and that the interaction of pERK with importin7 is greatly reduced in the Tm5NM1 KO cells.

scholarly journals Neuroprotective and Neurorestorative Effects of Holothuria scabra Extract in the MPTP/MPP+-Induced Mouse and Cellular Models of Parkinson’s Disease

2020 ◽  
Vol 14 ◽  
Author(s):  
Kunwadee Noonong ◽  
Prasert Sobhon ◽  
Morakot Sroyraya ◽  
Kulathida Chaithirayanon
Keyword(s):  
Dopaminergic Neurons ◽  
Substantia Nigra Pars Compacta ◽  
Motor Deficits ◽  
Holothuria Scabra ◽  
Cellular Models ◽  
Anti Oxidant ◽  
Pre Treatment ◽  
Induced Apoptosis

Extracts from Holothuria scabra (HS) have been shown to possess anti-inflammation, anti-oxidant and anti-cancer activities. More recently, it was shown to have neuroprotective potential in Caenorhabditis elegans PD model. Here, we assessed whether HS has neuroprotective and neurorestorative effects on dopaminergic neurons in both mouse and cellular models of PD. We found that both pre-treatment and post-treatment with HS improved motor deficits in PD mouse model induced with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) as determined by grid walk test. This was likely mediated by HS protective and restorative effects on maintaining the numbers of dopaminergic neurons and fibers in both substantia nigra pars compacta (SNpc) and striatum. In a cellular model of PD, HS significantly attenuated 1-methyl-4-phenylpyridinium (MPP+)-induced apoptosis of DAergic-like neurons differentiated from SH-SY5Y cells by enhancing the expression of Bcl-2, suppressing the expression of cleaved Caspase 3 and preventing depolarization of mitochondrial membrane. In addition, HS could stimulate the expression of tyrosine hydroxylase (TH) and suppressed the formation of α-synuclein protein. Taken together, our in vivo and in vitro findings suggested that HS is an attractive candidate for the neuroprotection rather than neurorestoration in PD.

Regulation of scute function by extramacrochaete in vitro and in vivo

Development ◽  
1994 ◽  
Vol 120 (12) ◽  
pp. 3595-3603 ◽  
Author(s):  
C.V. Cabrera ◽  
M.C. Alonso ◽  
H. Huikeshoven
Keyword(s):  
Expression Patterns ◽  
Transcription Activation ◽  
Loss Of Function ◽  
Wild Type ◽  
Helix Loop Helix ◽  
In The Wild ◽  
Yeast Assay ◽  
Number Of Cells

The pattern of adult sensilla in Drosophila is established by the dosage-sensitive interaction of two antagonistic groups of genes. Sensilla development is promoted by members of the achaete-scute complex and the daughterless gene whereas it is suppressed by whereas extramacrochaete (emc) and hairy. All these genes encode helix-loop-helix proteins. The products of the achaete-scute complex and daughterless interact to form heterodimers able to activate transcription. In this report, we show that (1) extra-macrochaete forms heterodimers with the achaete, scute, lethal of scute and daughterless products; (2) extramacrochaete inhibits DNA-binding of Achaete, Scute and Lethal of Scute/Daughterless heterodimers and Daughterless homodimers and (3) extramacrochaete inhibits transcription activation by heterodimers in a yeast assay system. In addition, we have studied the expression patterns of scute in wild-type and extramacrochaete mutant imaginal discs. Expression of scute RNA during imaginal development occurs in groups of cells, but high levels of protein accumulate in the nuclei of only a subset of the RNA-expressing cells. The pattern is dynamic and results in a small number of protein-containing cells that correspond to sensillum precursors. extramacrochaete loss-of-function alleles develop extra sensilla and correspondingly display a larger number of cells with scute protein. These cells appear to arise from those that in the wild type already express scute RNA; hence, extramacrochaete is a repressor of scute function whose action may take place post-transcriptionally.

Knockout of the PKC Substrate Pleckstrin Causes Pleomorphic Defects in Platelets, Lymphocytes and Granulocytes.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 394-394
Author(s):  
Lurong Lian ◽  
Yanfeng Wang ◽  
Xinsheng Chen ◽  
Tami Bach ◽  
Laurie Lenox ◽  
...  
Keyword(s):  
T Cells ◽  
Platelet Aggregation ◽  
Alternative Pathway ◽  
Second Messengers ◽  
Pi3k Inhibitor ◽  
Loss Of Function ◽  
Wild Type

Abstract Pleckstrin is a 40 kDa phosphoprotein containing amino- and carboxyl-terminal Pleckstrin Homology (PH) domains separated by a DEP domain. Pleckstrin’s expression is restricted to platelets and leukocytes, and represents approximately 1% of total cellular protein within these cells. Following platelet and leukocyte activation, PKC rapidly phosphorylates pleckstrin inducing it to bind membrane bound phospholipids such as phosphatidylinositol 4,5 bisphosphate (PIP2). Heterologously expressed phosphorylated pleckstrin colocalized with integrins and induces cytoskeletal reorganization. To better define the role of pleckstrin in vivo, we introduced a loss-of-function mutation into the murine pleckstrin gene. Pleckstrin-null mice were present in offspring at a frequency consistent with a Mendelian inheritance pattern. Adult pleckstrin −/− mice had 32% lower platelet counts than their littermates, but exhibited no spontaneous hemorrhage. Given the role of PKC and phospholipid second messengers on cytoskeletal dynamics, and our observations of pleckstrin overexpression in cell lines, we analyzed whether loss of pleckstrin affected cell spreading. Pleckstrin −/− platelets spread extremely poorly upon immobilized fibrinogen, and rarely exhibited broad membrane extensions. Granulocytes from pleckstrin −/− mice also have a spreading defect, as well as impaired ability to generate reactive oxygen species in the response to TNFα. Knockout B-cells, CD4-T-cells, and CD8-T-cells all migrated approximately 30% as efficiently as wild type cells in response to a gradient of SDF-1α in a transwell assay. These data suggest that loss of pleckstrin causes cytoskeletal defects in cells of multiple hematopoietic lineages. Analyzing whether this caused a functional defect, we found that pleckstrin −/− platelets exhibited a 22% dense- and 24% alpha-granule exocytosis defect, and a 35% defect in thrombin-induced calcium entry. In spite of these abnormalities, platelets changed shape and aggregated normally after stimulation with thrombin, ADP, or collagen in vitro. Pleckstrin knockout platelets did have a markedly impaired aggregation response following exposure to the PKC stimulant, PMA. This suggested that pleckstrin is a critical effector for PKC-mediated aggregation, but another pathway is able to compensate for this loss of pleckstrin following agonist stimulation. We reasoned that the alternative pathway might also utilize PIP2-dependent second messengers. Since the phosphorylation of PIP2 by PI3K generates second messengers that also contribute to platelet aggregation, we tested whether PI3K compensated for the loss of pleckstrin. We found that the PI3K inhibitor, LY294002 profoundly impaired the aggregation of pleckstrin knockout platelets in response to stimulation of the thrombin receptor. In contrast, the PI3K inhibitor minimally affected wild type platelets. This demonstrates that second messengers generated by PI3K are able to compensate for loss of pleckstrin. This also demonstrates that thrombin-induced platelet aggregation can be mediated by one of two parallel pathways, one involving PKC and pleckstrin, and the other involving PI3K. Together, our results show that pleckstrin is an essential component of PKC-mediated platelet activation and signals directed to the cytoskeleton.

Sphingosine 1-phosphate stimulates eyelid closure in the developing rat by stimulating EGFR signaling

2018 ◽  
Vol 11 (553) ◽  
pp. eaat1470 ◽  
Author(s):  
Ganlan Bian ◽  
Caiyong Yu ◽  
Ling Liu ◽  
Chao Fang ◽  
Kun Chen ◽  
...  
Keyword(s):  
Nuclear Translocation ◽  
Mitogen Activated Protein Kinase ◽  
Loss Of Function ◽  
Egfr Signaling ◽  
Eyelid Closure ◽  
Egfr Signaling Pathway ◽  
Sphingosine 1 Phosphate ◽  
Developing Rat

In many mammals, the eyelids migrate over the eye and fuse during embryogenesis to protect the cornea from damage during birth and early life. Loss-of-function mutations affecting the epidermal growth factor receptor (EGFR) signaling pathway cause an eyes-open-at-birth (EOB) phenotype in rodents. We identified an insertional mutation in Spinster homolog 2 (Spns2) in a strain of transgenic rats exhibiting the EOB phenotype. Spns2, a sphingosine 1-phosphate (S1P) transporter that releases S1P from cells, was enriched at the tip of developing eyelids in wild-type rat embryos. Spns2 expression or treatment with S1P or any one of several EGFR ligands rescued the EOB Spns2 mutant phenotype in vivo and in tissue explants in vitro and rescued the formation of stress fibers in primary keratinocytes from mutants. S1P signaled through the receptors S1PR1, S1PR2, and S1PR3 to activate extracellular signal–regulated kinase (ERK) and EGFR-dependent mitogen-activated protein kinase kinase kinase 1 (MEKK1)–c-Jun signaling. S1P also induced the nuclear translocation of the transcription factor MAL in a manner dependent on EGFR signaling. MAL and c-Jun stimulated the expression of the microRNAs miR-21 and miR-222, both of which target the metalloprotease inhibitor TIMP3, thus promoting metalloprotease activity. The metalloproteases ADAM10 and ADAM17 stimulated EGFR signaling by cleaving a membrane-anchored form of EGF to release the ligand. Our results outline a network by which S1P transactivates EGFR signaling through a complex mechanism involving feedback between several intra- and extracellular molecules to promote eyelid fusion in the developing rat.

scholarly journals Investigation of the in vitro and in vivo activity of the type IV pilus extension ATPase BfpD

2020 ◽  
Author(s):  
Jinlei Zhao ◽  
Shahista Nisa ◽  
Michael S. Donnenberg
Keyword(s):  
Atpase Activity ◽  
Loss Of Function ◽  
Wild Type ◽  
Multifunctional Protein ◽  
Mutant Proteins ◽  
E Coli ◽  
Type Iv ◽  
Kinetics Of

AbstractType IV pili (T4Ps) are multifunctional protein fibers found in many bacteria and archaea. All T4P systems have an extension ATPase, which provides the energy required to push structural subunits out of the membrane. We previously reported that the BfpD T4P ATPase from enteropathogenic E. coli (EPEC) has the expected hexameric structure and ATPase activity, the latter enhanced by the presence of the N-terminal cytoplasmic domains of its partner proteins BfpC and BfpE. In this study, we further investigated the kinetics of the BfpD ATPase. Despite high purity of the proteins, the reported enhanced ATPase activity was found to be from (an) ATPase(s) contaminating the N-BfpC preparation. Furthermore, although two mutations in highly conserved bfpD sites led to loss of function in vivo, the purified mutant proteins retained some ATPase activity, albeit less than the wild-type protein. Therefore, the observed ATPase activity of BfpD was also affected by (a) contaminating ATPase(s). Expression of the mutant bfpD alleles did not interfere with BfpD function in bacteria that also expressed wild-type BfpD. However, a similar mutation of bfpF, which encodes the retraction ATPase, blocked the function of wild-type BfpF when both were present. These results highlight similarities and differences in function and activity of T4P extension and retraction ATPases in EPEC.

scholarly journals A novel NF-κB regulator encoded by circPLCE1 inhibits colorectal carcinoma progression by promoting RPS3 ubiquitin-dependent degradation

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Zhen-xing Liang ◽  
Hua-shan Liu ◽  
Li Xiong ◽  
Xin Yang ◽  
Feng-wei Wang ◽  
...  
Keyword(s):  
Colorectal Carcinoma ◽  
Cell Lines ◽  
Nuclear Translocation ◽  
Nuclear Factor Κb ◽  
Epigenetic Mechanism ◽  
Circular Rnas ◽  
Loss Of Function ◽  
Novel Protein

Abstract Background Constitutive activation of nuclear factor-κB (NF-κB) signaling plays a key role in the development and progression of colorectal carcinoma (CRC). However, the underlying mechanisms of excessive activation of NF-κB signaling remain largely unknown. Methods We used high throughput RNA sequencing to identify differentially expressed circular RNAs (circRNAs) between normal human intestinal epithelial cell lines and CRC cell lines. The identification of protein encoded by circPLCE1 was performed using LC–MS. The function of novel protein was validated in vitro and in vivo by gain or loss of function assays. Mechanistic results were concluded by immunoprecipitation analyses. Results A novel protein circPLCE1-411 encoded by circular RNA circPLCE1 was identified as a crucial player in the NF-κB activation of CRC. Mechanistically, circPLCE1-411 promoted the ubiquitin-dependent degradation of the critical NF-κB regulator RPS3 via directly binding the HSP90α/RPS3 complex to facilitate the dissociation of RPS3 from the complex, thereby reducing NF-κB nuclear translocation in CRC cells. Functionally, circPLCE1 inhibited tumor proliferation and metastasis in CRC cells, as well as patient-derived xenograft and orthotopic xenograft tumor models. Clinically, circPLCE1 was downregulated in CRC tissues and correlated with advanced clinical stages and poor survival. Conclusions circPLCE1 presents an epigenetic mechanism which disrupts NF-κB nuclear translocation and serves as a novel and promising therapeutic target and prognostic marker.

scholarly journals DAO1 catalyzes temporal and tissue-specific oxidative inactivation of auxin in Arabidopsis thaliana

2016 ◽  
Vol 113 (39) ◽  
pp. 11010-11015 ◽  
Author(s):  
Jun Zhang ◽  
Jinshan Ella Lin ◽  
Chinchu Harris ◽  
Fernanda Campos Mastrotti Pereira ◽  
Fan Wu ◽  
...  
Keyword(s):  
Acetic Acid ◽  
Plant Growth ◽  
Expression Patterns ◽  
Loss Of Function ◽  
Wild Type ◽  
Iaa Oxidase ◽  
Oxidative Inactivation ◽  
Mutant Phenotypes

Tight homeostatic regulation of the phytohormone auxin [indole-3-acetic acid (IAA)] is essential to plant growth. Auxin biosynthetic pathways and the processes that inactivate auxin by conjugation to amino acids and sugars have been thoroughly characterized. However, the enzyme that catalyzes oxidation of IAA to its primary catabolite 2-oxindole-3-acetic acid (oxIAA) remains uncharacterized. Here, we show that DIOXYGENASE FOR AUXIN OXIDATION 1 (DAO1) catalyzes formation of oxIAA in vitro and in vivo and that this mechanism regulates auxin homeostasis and plant growth. Null dao1-1 mutants contain 95% less oxIAA compared with wild type, and complementation of dao1 restores wild-type oxIAA levels, indicating that DAO1 is the primary IAA oxidase in seedlings. Furthermore, dao1 loss of function plants have altered morphology, including larger cotyledons, increased lateral root density, delayed sepal opening, elongated pistils, and reduced fertility in the primary inflorescence stem. These phenotypes are tightly correlated with DAO1 spatiotemporal expression patterns as shown by DAO1pro:β-glucuronidase (GUS) activity and DAO1pro:YFP-DAO1 signals, and transformation with DAO1pro:YFP-DAO1 complemented the mutant phenotypes. The dominant dao1-2D mutant has increased oxIAA levels and decreased stature with shorter leaves and inflorescence stems, thus supporting DAO1 IAA oxidase function in vivo. A second isoform, DAO2, is very weakly expressed in seedling root apices. Together, these data confirm that IAA oxidation by DAO1 is the principal auxin catabolic process in Arabidopsis and that localized IAA oxidation plays a role in plant morphogenesis.

Critical Role of CIB1 in Endothelial Cell Function and In Vivo Ischemia-Induced Angiogenesis.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1800-1800
Author(s):  
Mohamed A. Zayed ◽  
Andrew McFadden ◽  
Weiping Yuan ◽  
Mary E. Hartnett ◽  
Dan Chalothorn ◽  
...  
Keyword(s):  
Hind Limb ◽  
Ex Vivo ◽  
Critical Role ◽  
Tube Formation ◽  
Wild Type ◽  
Wild Type Littermate ◽  
Retinal Angiogenesis

Abstract CIB1, a 22kDa EF-hand containing calcium binding protein, was originally identified in a yeast two-hybrid screen as a binding partner for the cytoplasmic tail of the platelet integrin αIIb. CIB1 also associates with a number of kinases and modulates their activity, suggesting that CIB1 is an important regulatory molecule. Recently, we found that CIB1 is expressed in multiple endothelial cell (EC) types. We therefore tested the role of CIB1 in EC function in vitro, and in angiogenesis both ex vivo and in vivo. To test the role of CIB1 in EC function in vitro, we reduced endogenous CIB1 levels in ECs by RNA interference with an shRNA-delivered by lentivirus. CIB1 depletion significantly decreased EC haptotaxis on fibronectin and EC vascular tube formation on growth factor-reduced Matrigel. Treatment with FGF-2, an angiogenic factor, did not counter the observed inhibition of haptotaxis and tube formation by shRNA against CIB1. However, CIB1 overexpression enhanced FGF-2-induced EC haptotaxis relative to control cells. Similarly, ECs derived from CIB1 null mice exhibited a significant decrease in haptotaxis, tube formation, and proliferation compared to ECs isolated from wild-type littermate controls. In ex vivo aortic ring and tibialis anterior muscle culture assays, CIB1 null cultures supplemented with serum or FGF-2 demonstrated reduced blood vessel sprouting compared to wild-type littermate control cultures. Finally, in vivo assays for hyperoxic retinal angiogenesis and hind-limb induced-ischemia revealed a decrease in post-ischemia retinal neovascularization and Doppler hind-limb blood perfusion recovery, although developmental retinal angiogenesis in CIB1 null mice appeared normal. In conclusion, these findings support a critical role for CIB1 in EC function that appears to be important for ischemia-induced angiogenesis.

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