scholarly journals Repurposing cell growth-regulating compounds identifies kenpaullone which ameliorates pathologic pain via normalization of inhibitory neurotransmission

2019 ◽  
Author(s):  
Michele Yeo ◽  
Yong Chen ◽  
Changyu Jiang ◽  
Gang Chen ◽  
Kaiyuan Wang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Growth ◽  
Cortical Neurons ◽  
Reversal Potential ◽  
Spinal Cord Dorsal Horn ◽  
Ion Concentration ◽  
Inhibitory Neurotransmission ◽  
Human Neurons ◽  
And Function

AbstractInhibitory GABA-ergic neurotransmission is fundamental for the adult vertebrate central nervous system and requires low chloride ion concentration in neurons. This basic ionic-homeostatic mechanism relies on expression and function of KCC2, a neuroprotective ionic transporter that extrudes neuronal chloride. Importantly, no other transporter can rescue KCC2 deficit, and attenuated expression of KCC2 is strongly associated with circuit malfunction in chronic pain, epilepsy, neuro-degeneration, neuro-trauma, and other neuro-psychiatric illnesses. To isolate Kcc2 gene expression-enhancing compounds, we screened 1057 cell growth-regulating compounds in cultured primary cortical neurons. We identified kenpaullone (KP), which enhanced Kcc2/KCC2 expression and function in cultured rodent and human neurons by inhibiting GSK3ß. KP effectively reduced pathologic pain in preclinical mouse models of nerve constriction injury and bone cancer. In nerve-injury pain, KP restored Kcc2 expression and GABA-evoked chloride reversal potential in the spinal cord dorsal horn. Delta-catenin, a phosphorylation-target of GSK3ß in neurons, activated the Kcc2 promoter via Kaiso transcription factor. Validating this new pathway in-vivo, transient spinal over-expression of delta-catenin mimicked KP analgesia. With relevance for pathologic pain, our discoveries of a newly repurposed compound and a novel genetically-encoded mechanism that each enhance Kcc2 gene expression enable us to re-normalize disrupted inhibitory neurotransmission through genetic re-programming.

scholarly journals Repurposing cancer drugs identifies kenpaullone which ameliorates pathologic pain in preclinical models via normalization of inhibitory neurotransmission

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Michele Yeo ◽  
Yong Chen ◽  
Changyu Jiang ◽  
Gang Chen ◽  
Kaiyuan Wang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Nerve Injury ◽  
Cortical Neurons ◽  
Chloride Concentration ◽  
Reversal Potential ◽  
Bone Cancer ◽  
Spinal Cord Dorsal Horn ◽  
Inhibitory Neurotransmission ◽  
Human Neurons ◽  
And Function

AbstractInhibitory GABA-ergic neurotransmission is fundamental for the adult vertebrate central nervous system and requires low chloride concentration in neurons, maintained by KCC2, a neuroprotective ion transporter that extrudes intracellular neuronal chloride. To identify Kcc2 gene expression‑enhancing compounds, we screened 1057 cell growth-regulating compounds in cultured primary cortical neurons. We identified kenpaullone (KP), which enhanced Kcc2/KCC2 expression and function in cultured rodent and human neurons by inhibiting GSK3ß. KP effectively reduced pathologic pain-like behavior in mouse models of nerve injury and bone cancer. In a nerve-injury pain model, KP restored Kcc2 expression and GABA-evoked chloride reversal potential in the spinal cord dorsal horn. Delta-catenin, a phosphorylation-target of GSK3ß in neurons, activated the Kcc2 promoter via KAISO transcription factor. Transient spinal over-expression of delta-catenin mimicked KP analgesia. Our findings of a newly repurposed compound and a novel, genetically-encoded mechanism that each enhance Kcc2 gene expression enable us to re-normalize disrupted inhibitory neurotransmission through genetic re-programming.

Possible Effects of Depolarizing GABAA Conductance on the Neuronal Input–Output Relationship: A Modeling Study

2005 ◽  
Vol 93 (6) ◽  
pp. 3504-3523 ◽  
Author(s):  
Kenji Morita ◽  
Kunichika Tsumoto ◽  
Kazuyuki Aihara
Keyword(s):  
Firing Rate ◽  
Cortical Neurons ◽  
Reversal Potential ◽  
Pyramidal Cells ◽  
Resting Potential ◽  
Input Output ◽  
Wide Range ◽  
The Relationship

Recent in vitro experiments revealed that the GABAA reversal potential is about 10 mV higher than the resting potential in mature mammalian neocortical pyramidal cells; thus GABAergic inputs could have facilitatory, rather than inhibitory, effects on action potential generation under certain conditions. However, how the relationship between excitatory input conductances and the output firing rate is modulated by such depolarizing GABAergic inputs under in vivo circumstances has not yet been understood. We examine herewith the input–output relationship in a simple conductance-based model of cortical neurons with the depolarized GABAA reversal potential, and show that a tonic depolarizing GABAergic conductance up to a certain amount does not change the relationship between a tonic glutamatergic driving conductance and the output firing rate, whereas a higher GABAergic conductance prevents spike generation. When the tonic glutamatergic and GABAergic conductances are replaced by in vivo–like highly fluctuating inputs, on the other hand, the effect of depolarizing GABAergic inputs on the input–output relationship critically depends on the degree of coincidence between glutamatergic input events and GABAergic ones. Although a wide range of depolarizing GABAergic inputs hardly changes the firing rate of a neuron driven by noncoincident glutamatergic inputs, a certain range of these inputs considerably decreases the firing rate if a large number of driving glutamatergic inputs are coincident with them. These results raise the possibility that the depolarized GABAA reversal potential is not a paradoxical mystery, but is instead a sophisticated device for discriminative firing rate modulation.

P0658MATRIX AND FLOW MODULATE GENE EXPRESSION IN A 3D VASCULARISED TUBULE MODEL

2020 ◽  
Vol 35 (Supplement_3) ◽  
Author(s):  
Julie Williams ◽  
Sanlin Robinson ◽  
Babak Alaei ◽  
Kimberly Homan ◽  
Maryam Clausen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Extracellular Matrix ◽  
Impact Analysis ◽  
Cell Types ◽  
Drug Uptake ◽  
Shear Stresses ◽  
The Matrix ◽  
And Function

Abstract Background and Aims Questions abound regarding the translation of in vitro 2D cell culture systems to the human setting. This is especially true of the kidney in which there is a complex hierarchical structure and a multitude of cell types. While it is well accepted that extracellular matrix plays a large part in directing cellular physiology emerging research has highlighted the importance of shear stresses and flow rates too. To fully recapitulate the normal gene expression and function of a particular renal cell type how important is it to completely reconstitute their in vivo surroundings? Method To answer this question, we have cultured proximal tubular (PT) epithelial cells in a 3-dimensional channel embedded within an engineered extracellular matrix (ECM) under physiological flow that is colocalised with an adjacent channel lined with renal microvascular endothelial cells that mimic a peritubular capillary. Modifications to the system were made to allow up to 12 chips to be run in parallel in an easily handleable form. After a period of maturation under continuous flow, both cell types were harvested for RNAseq analyses. RNA expression data was compared with cells cultured under static 2-dimensional conditions on plastic or the engineered ECM. Additionally, the perfusion of glucose through this 3D vascularised PT model has been investigated in the presence and absence of known diabetes modulating agents. Results PCA of RNAseq data showed that a) static non-coated, b) static matrix-coated and c) flow matrix-coated conditions separated into 3 distinct groups, while cell co-culture had less impact. Analysis of transcriptomic signatures showed that many genes were modulated by the matrix with additional genes influenced under flow conditions. Several of these genes, classified as transporters, are of particular importance when using this model to assess drug uptake and safety implications. Co-culture regulated some interesting genes, but fewer than anticipated. Preliminary experiments are underway to monitor glucose uptake and transport between tubules under different conditions. Conclusion We have developed a medium throughput system in which matrix and flow modulate gene expression. This system can be used to study the physiology of molecular cross-talk between cells. Ongoing analysis will further consider relevance to human physiology.

Novel Murine Model To Study Modulation of Genes and Molecular Pathways Induced Following In Vivo Interaction between Multiple Myeloma Cells and Human BM Milieu.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3409-3409
Author(s):  
Paola Neri ◽  
Pierfrancesco Tassone ◽  
Masood Shammas ◽  
Mariateresa Fulciniti ◽  
Yu-Tzu Tai ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Growth ◽  
Gene Expression Profile ◽  
Expression Profile ◽  
Murine Model ◽  
Cell Signalling

Abstract Interaction between multiple myeloma (MM) cells and the bone marrow (BM) microenvironment plays a critical role in promoting MM cell growth, survival, migration and development of drug resistance. This interaction within the bone marrow milieu is unique and its understanding is important in evaluating effects of novel agents in vitro and in vivo. We here describe a novel murine model that allows us to study the expression changes in vivo in MM cells within the human BM milieu. In this model, the green fluorescent protein (INA-6 GFP+) transduced IL-6-dependent human MM cell line, INA-6, was injected in human bone chip implanted into SCID mice. At different time points the bone chip was retrieved, cells flushed out and GFP+ MM cells were purified by CD138 MACS microbeads. Similar isolation process was used on INA-6 GFP+ cells cultured in vitro and used as control. Total RNA was isolated from these cells and gene expression profile analyzed using the HG-U133 array chip (Affymetrix) and DChip analyzer program. We have identified significant changes in expression of several genes following in vivo interaction between INA-6 and the BM microenvironment. Specifically, we observed up-regulation of genes associated with cytokines (IL-4, IL-8, IGFB 2–5) and chemokines (CCL2, 5, 6, 18, 24, CCR1, 2, 4), implicated in cell-cell signalling. Moreover genes implicated in DNA transcription (V-Fos, V-Jun, V-kit), adhesion (Integrin alpha 2b, 7, cadherin 1 and 11) and cell growth (CDC14, Cyclin G2, ADRA1A) were also up-regulated and genes involved in apoptosis and cell death (p-57, BCL2, TNF1a) were down-regulated. Using the Ingenuity Pathway Analysis the most relevant pathways modulated by the in vivo interaction between MM cells and BMSCs were IL-6, IGF1, TGF-beta and ERK/MAPK-mediated pathways as well as cell-cycle regulation and chemokine signalling. These results are consistent with previously observed in vitro cell signalling studies. Taken together these results highlight the ability of BM microenvironment to modulate the gene expression profile of the MM cells and our ability to in vivo monitor the changes. This model thus provides us with an ability to study in vivo effects of novel agents on expression profile of MM cells in BM milieu, to pre-clinically characterize their activity.

Biologic consequences of androgen receptor (AR) activity in MDV3100-resistant LNCaP cells and dependence on AR full length.

2014 ◽  
Vol 32 (4_suppl) ◽  
pp. 74-74
Author(s):  
Yoshiaki Yamamoto ◽  
Yohann Loriot ◽  
Eliana Beraldi ◽  
Tianyuan Zhou ◽  
Youngsoo Kim ◽  
...  
Keyword(s):  
Gene Expression ◽  
Androgen Receptor ◽  
Cell Growth ◽  
Functional Role ◽  
Full Length ◽  
Lncap Cells ◽  
Exon 1

74 Background: While recent reports link androgen receptor (AR) variants (AR-Vs) to castration resistant prostate cancer (CRPC), the biological significance of AR-Vs in AR-regulated cell survival and proliferation, independent of AR full length (AR-FL), remains controversial. To define the functional role of AR-FL and AR-Vs in MDV3100-resistant (MDV-R), we designed antisense oligonucleotide (ASO) targeting exon 1 and exon 8 in AR to knockdown AR-FL alone or in combination with AR-Vs and examined these effects in MDV-R LNCaP-derived cells in vitro and in vivo. Methods: We generated by selection MDV-R LNCaP-derived sub-lines that uniformly expressed high levels of both AR-FL and AR-V7 compared to CRPC LNCaP xenografts. Cell growth rates, protein and gene expression were analyzed using crystal violet assay, western blotting and real-time PCR, respectively. Exon 1 and 8 AR-ASO were evaluated in MDV-R49F CRPC LNCaP xenografts. Results: AR-V7 was transiently transfected in MDV-R49F cells and differential knockdown of AR-V7 and/or AR-FL by exon 1 versus exon 8 AR-ASO was used to evaluate relative biologic contributions of AR-FL versus AR-V7 in MDV-R LNCaP AR-V7 overexpressing cells. Exon 1 and 8 AR-ASO treatment in these cells similarly decreased prostate-specific antigen (PSA) expression and induced apoptosis as measured by caspase-3 and PARP cleavage and cell growth inhibition. To further define the functional role of AR-Vs in MDV-R LNCaP cells, we used a CE3 siRNA that specifically silenced AR-V7, but not AR-FL in MDV-R LNCaP cells. AR-V7 knockdown did not decrease PSA levels, did not induce apoptosis, and did not inhibit cell growth. In MDV-R LNCaP cells, exon 1 and 8 ASO similarly suppressed cell growth and AR-regulated gene expression in vitro and in vivo. Conclusions: These results indicate that the AR remains an important driver of MDV3100 resistance and, the biologic consequences mainly driven by AR-FL in MDV-R LNCaP models.

scholarly journals Suppressive effects of dehydroepiandrosterone and the nuclear factor-κB inhibitor parthenolide on corticotroph tumor cell growth and function in vitro and in vivo

2006 ◽  
Vol 188 (2) ◽  
pp. 321-331 ◽  
Author(s):  
T Taguchi ◽  
T Takao ◽  
Y Iwasaki ◽  
M Nishiyama ◽  
K Asaba ◽  
...  
Keyword(s):  
Cell Growth ◽  
Tumor Growth ◽  
Tumor Cells ◽  
Survivin Expression ◽  
Nuclear Factor Κb ◽  
Nuclear Factor ◽  
Aging Effects ◽  
And Function

Dehydroepiandrosterone (DHEA) is believed to have an anti-tumor effect, as well as anti-inflammatory, antioxidant, and anti-aging effects. To clarify the possible inhibitory action of DHEA on pituitary tumor cells, we tested the effects of DHEA, alone or in combination with the nuclear factor-κB (NF-κB) inhibitor parthenolide (PRT), on AtT20 corticotroph cell growth and function both in vitro and in vivo. We found that, in vitro, DHEA and PRT had potent inhibitory effects on pro-opiomelanocortin and NF-κB-dependent gene expression. They also suppressed the transcription activity of survivin, a representative anti-apoptotic factor, and induced apoptosis in this cell line. Furthermore, using BALB/C nude mice with xenografts of AtT20 cells in vivo, we found that the combined administration of DHEA and PRT significantly attenuated tumor growth and survivin expression. The treatment also decreased the elevated plasma corticosterone levels and ameliorated the malnutrition induced by tumor growth. Altogether, these results suggested that combined treatments of DHEA and PRT potently inhibit the growth and function of corticotroph tumor cells both in vitro and in vivo. This effect may, at least partly, be caused by the suppressive effects of these compounds, such as survivin and other inhibitor of apoptosis proteins, on NF-κB-mediated gene transcription.

scholarly journals An IRF4-MYC-mTORC1 integrated pathway controls cell growth and the proliferative capacity of activated B cells during B cell differentiation in vivo

2021 ◽  
Author(s):  
Dillon G Patterson ◽  
Anna K Kania ◽  
Madeline J Price ◽  
James R Rose ◽  
Christopher D Scharer ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Differentiation ◽  
B Cells ◽  
Cell Growth ◽  
B Cell ◽  
Proliferative Response ◽  
Cell Activation ◽  
B Cell Differentiation ◽  
B Cell Activation

Cell division is an essential component of B cell differentiation to antibody-secreting plasma cells, with critical reprogramming occurring during the initial stages of B cell activation. However, a complete understanding of the factors that coordinate early reprogramming events in vivo remain to be determined. In this study, we examined the initial reprogramming by IRF4 in activated B cells using an adoptive transfer system and mice with a B cell-specific deletion of IRF4. IRF4-deficient B cells responding to influenza, NP-Ficoll and LPS divided, but stalled during the proliferative response. Gene expression profiling of IRF4-deficient B cells at discrete divisions revealed IRF4 was critical for inducing MYC target genes, oxidative phosphorylation, and glycolysis. Moreover, IRF4-deficient B cells maintained an inflammatory gene expression signature. Complementary chromatin accessibility analyses established a hierarchy of IRF4 activity and identified networks of dysregulated transcription factor families in IRF4-deficient B cells, including E-box binding bHLH family members. Indeed, B cells lacking IRF4 failed to fully induce Myc after stimulation and displayed aberrant cell cycle distribution. Furthermore, IRF4-deficient B cells showed reduced mTORC1 activity and failed to initiate the B cell-activation unfolded protein response and grow in cell size. Myc overexpression in IRF4-deficient was sufficient to overcome the cell growth defect. Together, these data reveal an IRF4-MYC-mTORC1 relationship critical for controlling cell growth and the proliferative response during B cell differentiation.

scholarly journals TRAF6 directs Foxp3 localization and facilitates Treg function through K63-type ubiquitination

2018 ◽  
Author(s):  
Xuhao Ni ◽  
Jinhui Tao ◽  
Jian Gu ◽  
Benjamin V. Park ◽  
Zuojia Chen ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
Transcription Factor ◽  
Transcriptional Regulation ◽  
Tumor Immunity ◽  
Immune Control ◽  
Treg Function ◽  
And Function ◽  
Expression And Activity

AbstractRegulatory T cells (Treg) are crucial mediators of immune control. The characteristic gene expression and suppressive function of Treg depend considerably on the stable expression and activity of the transcription factor Foxp3. While transcriptional regulation of the Foxp3 gene has been studied in depth, both the expression and function of Foxp3 are also modulated at the protein level. However, the molecular players involved in posttranslational Foxp3 regulation are just beginning to be elucidated. Here we found TRAF6-deficient Tregs were dysfunctional in vivo; mice with Treg-restricted deletion of TRAF6 were resistant to B16 melanomas and displayed enhanced anti-tumor immunity. We further determined that Foxp3 undergoes lysine-63 chain (K63) ubiquitination at lysine 262 mediated by the E3 ligase TRAF6. When deprived of TRAF6 activity or rendered insensitive to K63 ubiquitination, Foxp3 displayed aberrant, perinuclear accumulation, disrupted function. Thus, Foxp3 ubiquitination by TRAF6 ensures proper localization of Foxp3 and facilitates Foxp3’s gene-regulating activity in Tregs. These results implicate TRAF6 as a key posttranslational, Treg-stabilizing force that may be targeted in novel tolerance-breaking therapies.

scholarly journals Tendon-derived biomimetic surface topographies induce phenotypic maintenance of tenocytes in vitro

2020 ◽  
Author(s):  
Aysegul Dede Eren ◽  
Aliaksei Vasilevich ◽  
E. Deniz Eren ◽  
Phanikrishna Sudarsanam ◽  
Urandelger Tuvshindorj ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Shape ◽  
Flat Surface ◽  
Flat Surfaces ◽  
Tissue Culture Polystyrene ◽  
Surface Topographies ◽  
And Function ◽  
Shape Changes

AbstractThe tenocyte niche contains biochemical and biophysical signals that are needed for tendon homeostasis. The tenocyte phenotype is correlated with cell shape in vivo and in vitro, and shape-modifying cues are needed for tenocyte phenotypical maintenance. Indeed, cell shape changes from elongated to spread when cultured on a flat surface, and rat tenocytes lose the expression of phenotypical markers throughout five passages. We hypothesized that tendon gene expression can be preserved by culturing cells in the native tendon shape. To this end, we reproduced the tendon topographical landscape into tissue culture polystyrene, using imprinting technology. We confirmed that the imprints forced the cells into a more elongated shape, which correlated with the level of Scleraxis expression. When we cultured the tenocytes for seven days on flat surfaces and tendon imprints, we observed a decline in tenogenic marker expression on flat but not on imprints. This research demonstrates that native tendon topography is an important factor contributing to the tenocyte phenotype. Tendon imprints therefore provide a powerful platform to explore the effect of instructive cues originating from native tendon topography on guiding cell shape, phenotype and function of tendon-related cells.

scholarly journals Deficiency of intellectual disability-related gene Brpf1 reduced inhibitory neurotransmission and Map2k7 expression in GABAergic interneurons

2021 ◽  
Author(s):  
Jingli Cao ◽  
Weiwei Xian ◽  
Maierdan Palihati ◽  
Yu Zhu ◽  
Guoxiang Wang ◽  
...  
Keyword(s):  
Intellectual Disability ◽  
Dendritic Morphology ◽  
Gabaergic Interneurons ◽  
Related Gene ◽  
Inhibitory Neurotransmission ◽  
Whole Cell Patch Clamp ◽  
Developmental Niche ◽  
And Function

AbstractIntellectual disability is closely related to impaired GABA neurotransmission. Brpf1 was specifically expressed in medial ganglionic eminence (MGE), a developmental niche of GABAergic interneurons, and patients with BRPF1 mutations were mentally retarded. To test its role in development and function of MGE-derived GABAergic interneurons, we performed immunofluorescence staining, whole-cell patch-clamp, MGE transplantation and mRNA-Seq to understand its effect on neuronal differentiation, dendritic morphology, electrophysiology, migration and gene regulation, using mouse MGE-derived GABAergic interneurons infected with AAV-shBrpf1. We found a decreasing trend on parvalbumin+ interneuron differentiation. Moreover, increased firing threshold, decreased number of evoked APs, and a reduced amplitude of mIPSCs were observed before any significant change of MAP2+ dendritic morphology and in vivo migration appeared. Finally, mRNA-Seq analysis revealed that genes related to neurodevelopment and synaptic transmission such as Map2k7 were dysregulated. Our results demonstrated a key role of Brpf1 in inhibitory neurotransmission and related gene expression of GABAergic interneurons.

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