scholarly journals Interaction of Huntington Disease Protein with Transcriptional Activator Sp1

2002 ◽  
Vol 22 (5) ◽  
pp. 1277-1287 ◽  
Author(s):  
Shi-Hua Li ◽  
Anna L. Cheng ◽  
Hui Zhou ◽  
Suzanne Lam ◽  
Manjula Rao ◽  
...  
Keyword(s):  
Huntington Disease ◽  
Cultured Cells ◽  
Growth Factor Receptor ◽  
Soluble Form ◽  
Mutant Huntingtin ◽  
Nuclear Effect ◽  
Polyglutamine Expansion ◽  
Disease Protein ◽  
Cellular Dysfunction

ABSTRACT Polyglutamine expansion causes Huntington disease (HD) and at least seven other neurodegenerative diseases. In HD, N-terminal fragments of huntingtin with an expanded glutamine tract are able to aggregate and accumulate in the nucleus. Although intranuclear huntingtin affects the expression of numerous genes, the mechanism of this nuclear effect is unknown. Here we report that huntingtin interacts with Sp1, a transcription factor that binds to GC-rich elements in certain promoters and activates transcription of the corresponding genes. In vitro binding and immunoprecipitation assays show that polyglutamine expansion enhances the interaction of N-terminal huntingtin with Sp1. In HD transgenic mice (R6/2) that express N-terminal-mutant huntingtin, Sp1 binds to the soluble form of mutant huntingtin but not to aggregated huntingtin. Mutant huntingtin inhibits the binding of nuclear Sp1 to the promoter of nerve growth factor receptor and suppresses its transcriptional activity in cultured cells. Overexpression of Sp1 reduces the cellular toxicity and neuritic extension defects caused by intranuclear mutant huntingtin. These findings suggest that the soluble form of mutant huntingtin in the nucleus may cause cellular dysfunction by binding to Sp1 and thus reducing the expression of Sp1-regulated genes.

scholarly journals Mutant Huntingtin stalls ribosomes and represses protein synthesis in a cellular model of Huntington disease

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Mehdi Eshraghi ◽  
Pabalu P. Karunadharma ◽  
Juliana Blin ◽  
Neelam Shahani ◽  
Emiliano P. Ricci ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Huntington Disease ◽  
Ribosomal Proteins ◽  
Mutant Huntingtin ◽  
Translation Elongation ◽  
Polyglutamine Expansion ◽  
Ribosome Stalling ◽  
Mrna Targets ◽  
Discernible Effect

AbstractThe polyglutamine expansion of huntingtin (mHTT) causes Huntington disease (HD) and neurodegeneration, but the mechanisms remain unclear. Here, we found that mHtt promotes ribosome stalling and suppresses protein synthesis in mouse HD striatal neuronal cells. Depletion of mHtt enhances protein synthesis and increases the speed of ribosomal translocation, while mHtt directly inhibits protein synthesis in vitro. Fmrp, a known regulator of ribosome stalling, is upregulated in HD, but its depletion has no discernible effect on protein synthesis or ribosome stalling in HD cells. We found interactions of ribosomal proteins and translating ribosomes with mHtt. High-resolution global ribosome footprint profiling (Ribo-Seq) and mRNA-Seq indicates a widespread shift in ribosome occupancy toward the 5′ and 3′ end and unique single-codon pauses on selected mRNA targets in HD cells, compared to controls. Thus, mHtt impedes ribosomal translocation during translation elongation, a mechanistic defect that can be exploited for HD therapeutics.

scholarly journals In vitro modeling of glioblastoma initiation using PDGF-AA and p53-null neural progenitors

2020 ◽  
Vol 22 (8) ◽  
pp. 1150-1161 ◽  
Author(s):  
Alexandra K Bohm ◽  
Jessica DePetro ◽  
Carmen E Binding ◽  
Amanda Gerber ◽  
Nicholas Chahley ◽  
...  
Keyword(s):  
Growth Factor ◽  
Genome Stability ◽  
Cultured Cells ◽  
Growth Factor Receptor ◽  
Platelet Derived Growth Factor ◽  
Early Event ◽  
Wild Type ◽  
Microscopic Appearance ◽  
Human Gbm

Abstract Background Imagining ways to prevent or treat glioblastoma (GBM) has been hindered by a lack of understanding of its pathogenesis. Although overexpression of platelet derived growth factor with two A-chains (PDGF-AA) may be an early event, critical details of the core biology of GBM are lacking. For example, existing PDGF-driven models replicate its microscopic appearance, but not its genomic architecture. Here we report a model that overcomes this barrier to authenticity. Methods Using a method developed to establish neural stem cell cultures, we investigated the effects of PDGF-AA on subventricular zone (SVZ) cells, one of the putative cells of origin of GBM. We microdissected SVZ tissue from p53-null and wild-type adult mice, cultured cells in media supplemented with PDGF-AA, and assessed cell viability, proliferation, genome stability, and tumorigenicity. Results Counterintuitive to its canonical role as a growth factor, we observed abrupt and massive cell death in PDGF-AA: wild-type cells did not survive, whereas a small fraction of null cells evaded apoptosis. Surviving null cells displayed attenuated proliferation accompanied by whole chromosome gains and losses. After approximately 100 days in PDGF-AA, cells suddenly proliferated rapidly, acquired growth factor independence, and became tumorigenic in immune-competent mice. Transformed cells had an oligodendrocyte precursor-like lineage marker profile, were resistant to platelet derived growth factor receptor alpha inhibition, and harbored highly abnormal karyotypes similar to human GBM. Conclusion This model associates genome instability in neural progenitor cells with chronic exposure to PDGF-AA and is the first to approximate the genomic landscape of human GBM and the first in which the earliest phases of the disease can be studied directly.

scholarly journals Proliferation and cytogenetic analysis of hairy cell leukemia upon stimulation via the CD40 antigen

Blood ◽  
1994 ◽  
Vol 84 (9) ◽  
pp. 3134-3141 ◽  
Author(s):  
HC Kluin-Nelemans ◽  
GC Beverstock ◽  
P Mollevanger ◽  
HW Wessels ◽  
E Hoogendoorn ◽  
...  
Keyword(s):  
Hairy Cell Leukemia ◽  
Cultured Cells ◽  
Thymidine Incorporation ◽  
Interleukin 4 ◽  
Soluble Form ◽  
Cross Linking ◽  
Hairy Cell ◽  
Cell Leukemia ◽  
In Vitro Proliferation

Abstract Using the CD40 system, in vitro proliferation of hairy cell leukemia (HCL) was examined in 43 patients. In this culture system, cells were stimulated by interleukin-4 (IL-4) and anti-CD40 monoclonal antibodies (MoAbs) that were added in soluble form or were cross-linked via their Fc part using Fc gamma RII-transfected mouse fibroblast cells. Proliferation was induced and confirmed by 3H-thymidine incorporation in 14 cases and by the presence of metaphases in 42 cases. 3H-thymidine incorporation showed a heterogeneous pattern: cross-linking of anti- CD40 gave the highest proliferation in 8 cases; in 11 cases, stimulation with anti-CD40 MoAbs alone, without cross-linking also resulted in proliferation; the addition of IL-4 further enhanced 3H- thymidine incorporation in 5 cases, but suppressed this phenomenon in 5 other cases. The CD40 system proved to be very effective in obtaining cytogenetic data. With a success rate of 42 of 43 patients tested, we found clonal abnormalities in 8 cases (19%) and nonclonal abnormalities with involvement of one or two abnormal metaphases in another 7 cases. The chromosomes most frequently involved in the abnormal karyotypes, both structurally and numerically, were chromosomes 5, 7, and 14. By fluorescence-activated cell-sorting analysis of the cultured cells, and by immunophenotypic analysis of metaphase spreads, T-cell growth could be excluded and the HCL-lineage confirmed. Stimulation via the CD40 antigen is an excellent tool for growing hairy cell leukemia cells.

scholarly journals Rhes travels from cell to cell and transports Huntington disease protein via TNT-like protrusion

2019 ◽  
Vol 218 (6) ◽  
pp. 1972-1993 ◽  
Author(s):  
Manish Sharma ◽  
Srinivasa Subramaniam
Keyword(s):  
Huntington Disease ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Mutant Huntingtin ◽  
Tunneling Nanotubes ◽  
Neighboring Cell ◽  
Disease Protein ◽  
Electron Microscopy Analysis ◽  
Microscopy Analysis ◽  
Cell Cell

Tunneling nanotubes (TNT) are thin, membranous, tunnel-like cell-to-cell connections, but the mechanisms underlying their biogenesis or functional role remains obscure. Here, we report, Rhes, a brain-enriched GTPase/SUMO E3-like protein, induces the biogenesis of TNT-like cellular protrusions, “Rhes tunnels,” through which Rhes moves from cell to cell and transports Huntington disease (HD) protein, the poly-Q expanded mutant Huntingtin (mHTT). The formation of TNT-like Rhes tunnels requires the Rhes’s serine 33, C-terminal CAAX, and a SUMO E3-like domain. Electron microscopy analysis revealed that TNT-like Rhes tunnels appear continuous, cell–cell connections, and <200 nm in diameter. Live-cell imaging shows that Rhes tunnels establish contact with the neighboring cell and deliver Rhes-positive cargoes, which travel across the plasma membrane of the neighboring cell before entering it. The Rhes tunnels carry Rab5a/Lyso 20-positive vesicles and transport mHTT, but not normal HTT, mTOR, or wtTau proteins. SUMOylation-defective mHTT, Rhes C263S (cannot SUMOylate mHTT), or CRISPR/Cas9-mediated depletion of three isoforms of SUMO diminishes Rhes-mediated mHTT transport. Thus, Rhes promotes the biogenesis of TNT-like cellular protrusions and facilitates the cell–cell transport of mHTT involving SUMO-mediated mechanisms.

scholarly journals The Ubiquitin-Proteasome System in Huntington’s Disease: Are Proteasomes Impaired, Initiators of Disease, or Coming to the Rescue?

2012 ◽  
Vol 2012 ◽  
pp. 1-12 ◽  
Author(s):  
Sabine Schipper-Krom ◽  
Katrin Juenemann ◽  
Eric A. J. Reits
Keyword(s):  
Huntington's Disease ◽  
Huntington’S Disease ◽  
Protein A ◽  
Ubiquitin Proteasome System ◽  
Mutant Huntingtin ◽  
Polyglutamine Expansion ◽  
Ubiquitin Proteasome ◽  
Intracellular Aggregates ◽  
Polyglutamine Aggregation

Huntington’s disease is a progressive neurodegenerative disease, caused by a polyglutamine expansion in the huntingtin protein. A prominent hallmark of the disease is the presence of intracellular aggregates initiated by N-terminal huntingtin fragments containing the polyglutamine repeat, which recruit components of the ubiquitin-proteasome system. While it is commonly thought that proteasomes are irreversibly sequestered into these aggregates leading to impairment of the ubiquitin-proteasome system, the data on proteasomal impairment in Huntington’s disease is contradictory. In addition, it has been suggested that proteasomes are unable to actually cleave polyglutamine sequencesin vitro, thereby releasing aggregation-prone polyglutamine peptides in cells. Here, we discuss how the proteasome is involved in the various stages of polyglutamine aggregation in Huntington’s disease, and how alterations in activity may improve clearance of mutant huntingtin fragments.

Megalomicin disrupts lysosomal functions

1997 ◽  
Vol 110 (16) ◽  
pp. 1839-1849
Author(s):  
P. Bonay ◽  
M. Fresno ◽  
B. Alarcon
Keyword(s):  
Epidermal Growth Factor Receptor ◽  
Protein Transport ◽  
Cultured Cells ◽  
Growth Factor Receptor ◽  
Fluid Phase ◽  
Epidermal Growth ◽  
Membrane Bound ◽  
Sialylated Glycoproteins

Megalomicin (MGM) has been shown to cause a dilation of the most distal cisternae of the Golgi complex. The effects of MGM on Golgi morphology correlated with an inhibition of protein transport to the trans-Golgi resulting in an accumulation of poorly sialylated glycoproteins. Here we show that the addition of 50 microM MGM caused a rapid swelling of lysosomes in cultured cells and inhibited the degradation of the newly synthesized T cell antigen receptor CD36 subunit. Although MGM did not affect the uptake of fluid phase markers, it prevented their degradation. Interestingly, endocytosed ovalbumin did not colocalize with lysosomes in MGM-treated cells, suggesting an MGM-induced impairment in the delivery to lysosomes. This was confirmed by Percoll density gradients, where the fluid phase marker remained in endosomal fractions, even after long chase times, whereas in control cells the endocytosed marker was located in lysosomes. The effect of MGM was not confined to soluble proteins since it did also inhibit the delivery of the membrane-bound epidermal growth factor receptor to lysosomes. Finally, MGM strongly inhibited the ATP-dependent acidification of lysosomes in vitro, suggesting a possible mechanism for its in vivo activity.

scholarly journals A novel variant of FGFR3 causes proportionate short stature

2015 ◽  
Vol 172 (6) ◽  
pp. 763-770 ◽  
Author(s):  
Sarina G Kant ◽  
Iveta Cervenkova ◽  
Lukas Balek ◽  
Lukas Trantirek ◽  
Gijs W E Santen ◽  
...  
Keyword(s):  
Short Stature ◽  
Exome Sequencing ◽  
Rare Variant ◽  
Sanger Sequencing ◽  
Cultured Cells ◽  
Diagnostic Testing ◽  
Growth Factor Receptor ◽  
Three Dimensional Model ◽  
Functional Studies

ObjectiveMutations of the fibroblast growth factor receptor 3 (FGFR3) cause various forms of short stature, of which the least severe phenotype is hypochondroplasia, mainly characterized by disproportionate short stature. Testing for an FGFR3 mutation is currently not part of routine diagnostic testing in children with short stature without disproportion.DesignA three-generation family A with dominantly transmitted proportionate short stature was studied by whole-exome sequencing to identify the causal gene mutation. Functional studies and protein modeling studies were performed to confirm the pathogenicity of the mutation found in FGFR3. We performed Sanger sequencing in a second family B with dominant proportionate short stature and identified a rare variant in FGFR3.MethodsExome sequencing and/or Sanger sequencing was performed, followed by functional studies using transfection of the mutant FGFR3 into cultured cells; homology modeling was used to construct a three-dimensional model of the two FGFR3 variants.ResultsA novel p.M528I mutation in FGFR3 was detected in family A, which segregates with short stature and proved to be activating in vitro. In family B, a rare variant (p.F384L) was found in FGFR3, which did not segregate with short stature and showed normal functionality in vitro compared with WT.ConclusionsProportionate short stature can be caused by a mutation in FGFR3. Sequencing of this gene can be considered in patients with short stature, especially when there is an autosomal dominant pattern of inheritance. However, functional studies and segregation studies should be performed before concluding that a variant is pathogenic.

scholarly journals Proliferation and cytogenetic analysis of hairy cell leukemia upon stimulation via the CD40 antigen

Blood ◽  
1994 ◽  
Vol 84 (9) ◽  
pp. 3134-3141 ◽  
Author(s):  
HC Kluin-Nelemans ◽  
GC Beverstock ◽  
P Mollevanger ◽  
HW Wessels ◽  
E Hoogendoorn ◽  
...  
Keyword(s):  
Hairy Cell Leukemia ◽  
Cultured Cells ◽  
Thymidine Incorporation ◽  
Interleukin 4 ◽  
Soluble Form ◽  
Cross Linking ◽  
Hairy Cell ◽  
Cell Leukemia ◽  
In Vitro Proliferation

Using the CD40 system, in vitro proliferation of hairy cell leukemia (HCL) was examined in 43 patients. In this culture system, cells were stimulated by interleukin-4 (IL-4) and anti-CD40 monoclonal antibodies (MoAbs) that were added in soluble form or were cross-linked via their Fc part using Fc gamma RII-transfected mouse fibroblast cells. Proliferation was induced and confirmed by 3H-thymidine incorporation in 14 cases and by the presence of metaphases in 42 cases. 3H-thymidine incorporation showed a heterogeneous pattern: cross-linking of anti- CD40 gave the highest proliferation in 8 cases; in 11 cases, stimulation with anti-CD40 MoAbs alone, without cross-linking also resulted in proliferation; the addition of IL-4 further enhanced 3H- thymidine incorporation in 5 cases, but suppressed this phenomenon in 5 other cases. The CD40 system proved to be very effective in obtaining cytogenetic data. With a success rate of 42 of 43 patients tested, we found clonal abnormalities in 8 cases (19%) and nonclonal abnormalities with involvement of one or two abnormal metaphases in another 7 cases. The chromosomes most frequently involved in the abnormal karyotypes, both structurally and numerically, were chromosomes 5, 7, and 14. By fluorescence-activated cell-sorting analysis of the cultured cells, and by immunophenotypic analysis of metaphase spreads, T-cell growth could be excluded and the HCL-lineage confirmed. Stimulation via the CD40 antigen is an excellent tool for growing hairy cell leukemia cells.

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.

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