Abstract TP90: Casein Kinase 2 Inhibition Preserves Axonal Function Against Ischemia

Stroke ◽  
2017 ◽  
Vol 48 (suppl_1) ◽  
Author(s):  
Sylvain Brunet ◽  
Chinthasagar Bastian ◽  
Danielle Aquila ◽  
Selva Baltan
Keyword(s):  
White Matter ◽  
Ischemic Injury ◽  
Glucose Deprivation ◽  
Casein Kinase ◽  
Casein Kinase 2 ◽  
Mortality And Morbidity ◽  
Artificial Cerebrospinal Fluid ◽  
Compound Action Potentials ◽  
The Impact ◽  
Ck2 Inhibitors

Axonal injury and dysfunction are responsible for much of the disability observed following a stroke. Human brain comprises equal proportions of gray matter and white matter and white matter is injured in most strokes. Casein kinase 2 (CK2) is a protein kinase expressed in brain, including white matter, and is regulated by ischemia. We therefore hypothesized that transient CK2 inhibition would protect white matter from ischemic injury. To assess the impact of CK2 inhibition on axonal electrical activity following oxygen glucose deprivation (OGD), mouse optic nerves (MONs), a pure white matter track, from C57BL/6J were subjected to OGD (1h) while eliciting compound action potentials (CAPs) and exposed to CX-4945, a selective CK2 inhibitor, or control artificial cerebrospinal fluid (ACSF). We observed that CX-4945 preserved CAPs when applied either before or after OGD. Then to determine the impact of CK2 inhibition on glial cell survival following OGD, MONs exposed to OGD that were treated with either CX-4945 or control ACSF were processed for immunohistochemistry. We observed that CX-4945 treatment protected oligodendrocytes from OGD. And finally, to determine if CK2 inhibition protected mitochondrial from OGD, MONs from Thy-1 mito-CFP mice were similarly subjected to OGD in the presence of either CX-4945 or control ACSF. We observed that CX-4945 maintained Thy-1 mito-CFP fluorescence following OGD. In conclusion, our results suggest that CK2 inhibition preserves axonal function by preserving oligodendrocytes and mitochondrial function following ischemic injury. We propose that CK2 inhibitors, which are currently in phase II-III clinical trials for cancer therapy could be repurposed and provide a novel therapeutic target to protect white matter against ischemic injury, reducing mortality and morbidity and improving recovery following stroke.

scholarly journals Excitotoxic Mechanisms of Ischemic Injury in Myelinated White Matter

2007 ◽  
Vol 27 (9) ◽  
pp. 1540-1552 ◽  
Author(s):  
Selva Baltan Tekkök ◽  
ZuCheng Ye ◽  
Bruce R Ransom
Keyword(s):  
White Matter ◽  
High Performance ◽  
Glutamate Release ◽  
Glutamate Transporter ◽  
Ischemic Injury ◽  
Glucose Deprivation ◽  
Kainate Receptors ◽  
Adult Mice ◽  
Direct Exposure ◽  
Glutamate Receptor Agonist

Axonal injury and dysfunction in white matter (WM) are caused by many neurologic diseases including ischemia. We characterized ischemic injury and the role of glutamate-mediated excitotoxicity in a purely myelinated WM tract, the mouse optic nerve (MON). For the first time, excitotoxic WM injury was directly correlated with glutamate release. Oxygen and glucose deprivation (OGD) caused duration-dependent loss of axon function in optic nerves from young adult mice. Protection of axon function required blockade of both α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and kainate receptors, or removal of extracellular Ca2+. Blockade of N-methyl-D-aspartate receptors did not preserve axon function. Curiously, even extended periods of direct exposure to glutamate or kainate or AMPA failed to induce axon dysfunction. Brief periods of OGD, however, caused glutamate receptor agonist exposure to become toxic, suggesting that ionic disruption enabled excitotoxic injury. Glutamate release, directly measured using quantitative high-performance liquid chromatography, occurred late during a 60-mins period of OGD and was due to reversal of the glutamate transporter. Brief periods of OGD (i.e., 15 mins) did not cause glutamate release and produced minimal injury. These results suggested that toxic glutamate accumulation during OGD followed the initial ionic changes mediating early loss of excitability. The onset of glutamate release was an important threshold event for irreversible ischemic injury. Regional differences appear to exist in the specific glutamate receptors that mediate WM ischemic injury. Therapy for ischemic WM injury must be designed accordingly.

scholarly journals The Mechanisms of Acute Ischemic Injury in the Cell Processes of Developing White Matter Astrocytes

2007 ◽  
Vol 28 (3) ◽  
pp. 588-601 ◽  
Author(s):  
Michael G Salter ◽  
Robert Fern
Keyword(s):  
White Matter ◽  
Ischemic Injury ◽  
Glucose Deprivation ◽  
Oxygen Glucose Deprivation ◽  
Complete Protection ◽  
Cell Processes ◽  
The Central Nervous System ◽  
Ion Influx ◽  
Disulphonic Acid ◽  
Process Loss

Astrocytes are fundamentally important to the maintenance and proper functioning of the central nervous system. During the period of development when myelination is occurring, white matter astrocytes are particularly sensitive to ischemic injury and their failure to regulate glutamate during ischemic conditions may be an important factor in excitotoxic injury. Here, we have identified key mechanisms of injury that operate on the processes of immature white matter astrocytes during oxygen-glucose deprivation (OGD) using GFAP-GFP mice. Oxygen-glucose deprivation produced a parallel loss of astrocyte processes and somata, assessed by both the retention of GFP fluorescence within these structures and by quantitative electron microscopy. Oxygen-glucose deprivation-induced process loss was Ca2+ independent and had two distinct mechanisms. Substituting either extracellular Na+ or Cl−, or perfusion with the Na—K–Cl co-transport blocker bumetanide, provided protection up to 40 mins of OGD but not beyond that point. HCO−3 substitution or perfusion with 4,4′-diisothiocyanostilbene-2,2′-disulphonic acid provided complete protection of the processes up to 60 mins of OGD. Zero-Na+/zero-K+ conditions provided complete protection from OGD-induced injury of processes and somata at all time points. We conclude that acute ischemic-type injury of immature astrocytes follows a cytotoxic ion influx mediated in part by Na—K–Cl co-transport and in part by Na+- and K+-dependent HCO−3 transport, a mechanism that is common to both cell processes and somata. This work provides a basis on which preventative strategies may be developed to protect white matter astrocytes from ischemic injury in susceptible individuals.

scholarly journals Identification of miRNAs That Mediate Protective Functions of Anti-Cancer Drugs During White Matter Ischemic Injury

ASN NEURO ◽  
2021 ◽  
Vol 13 ◽  
pp. 175909142110422
Author(s):  
Selva Baltan ◽  
Ursula S. Sandau ◽  
Sylvain Brunet ◽  
Chinthasagar Bastian ◽  
Ajai Tripathi ◽  
...  
Keyword(s):  
White Matter ◽  
Mirna Expression ◽  
Expression Profiling ◽  
Ischemic Injury ◽  
Glucose Deprivation ◽  
Cancer Drugs ◽  
Protective Actions ◽  
Anti Cancer ◽  
Mirna Expression Profiling ◽  
Ck2 Inhibitor

We have previously shown that two anti-cancer drugs, CX-4945 and MS-275, protect and preserve white matter (WM) architecture and improve functional recovery in a model of WM ischemic injury. While both compounds promote recovery, CX-4945 is a selective Casein kinase 2 (CK2) inhibitor and MS-275 is a selective Class I histone deacetylase (HDAC) inhibitor. Alterations in microRNAs (miRNAs) mediate some of the protective actions of these drugs. In this study, we aimed to (1) identify miRNAs expressed in mouse optic nerves (MONs); (2) determine which miRNAs are regulated by oxygen glucose deprivation (OGD); and (3) determine the effects of CX-4945 and MS-275 treatment on miRNA expression. RNA isolated from MONs from control and OGD-treated animals with and without CX-4945 or MS-275 treatment were quantified using NanoString nCounter® miRNA expression profiling. Comparative analysis of experimental groups revealed that 12 miRNAs were expressed at high levels in MONs. OGD upregulated five miRNAs (miR-1959, miR-501-3p, miR-146b, miR-201, and miR-335-3p) and downregulated two miRNAs (miR-1937a and miR-1937b) compared to controls. OGD with CX-4945 upregulated miR-1937a and miR-1937b, and downregulated miR-501-3p, miR-200a, miR-1959, and miR-654-3p compared to OGD alone. OGD with MS-275 upregulated miR-2134, miR-2141, miR-2133, miR-34b-5p, miR-153, miR-487b, miR-376b, and downregulated miR-717, miR-190, miR-27a, miR-1959, miR-200a, miR-501-3p, and miR-200c compared to OGD alone. Interestingly, miR-501-3p and miR-1959 were the only miRNAs upregulated by OGD, and downregulated by OGD plus CX-4945 and MS-275. Therefore, we suggest that protective functions of CX-4945 or MS-275 against WM injury maybe mediated, in part, through miRNA expression.

Role of Casein Kinase 2 in Platelets Release from Megakaryocytes.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1534-1534
Author(s):  
Alieta Ciocea ◽  
Tatiana V. Byzova ◽  
Michael Kalafatis
Keyword(s):  
Flow Cytometry ◽  
Cell Line ◽  
Casein Kinase ◽  
Casein Kinase 2 ◽  
Myelogenous Leukemia ◽  
Coagulation Cascade ◽  
Binding Studies ◽  
Cytotoxic Treatment ◽  
Human Thrombin ◽  
Ck2 Inhibitors

Abstract Megakaryoblasts are precursors of platelets. Megakaryoblasts first differentiate to the stage of megakaryocytes. Megakaryocytes mature by increasing their size and by undergoing nuclear endoreplication and cytoplasmic maturation. Mature megakaryocytes form pseudopodia and reach pro-platelet bearing stage. Proplatelets bearing megakaryocytes fragment to give rise to platelets, through the process of thrombocytopoiesis. Both the thrombocytopoiesis and the megakaryocytopoiesis processes are linked to the constitutive apoptosis of megakaryocytic cells. The BCR/ABL oncoprotein is the result of a chromosomal translocation known as Philadelphia positive chromosome (Ph+). This is the main cause of several myeloproliferative disorders, like chronic myelogenous leukemia (CML). In CML the rate of platelet production is abnormal due to malignant megakaryoblast overproliferation. Casein kinase 2 (CK2) plays a crucial role in CML. CK2 was found to interact with BCR/ABL and to modulate its function. CK2 is also involved in many pathways downstream of BCR/ABL. CK2 acts on apoptotic pathways and also in the blood coagulation cascade. We studied the effect of casein kinase 2 alpha subunit (CK2α) inhibition in a megakaryoblastic cell line from a CML patient in blast crisis (MEG-01). We found that these inhibitors induce proliferation arrest while maintaining a steady cell number for a period of one week. Treated cells grew at a lower and constant rate than the non-treated ones, which are extremely prolific. Apoptosis of MEG-01 was induced by CK2 inhibitors, and this phenomenon was dose and time dependent. No necrosis was detected in the presence of the inhibitors, demonstrating that such compounds are not cytotoxic. Treatment of MEG-01 cells with CK2 inhibitors resulted in cells with apoptotic features like blebbing and specific apoptotic DNA fragmentation. Interestingly, these treatments lead to stimulation of megakaryocytopoiesis and thrombocytopoiesis processes in MEG-01 megakaryoblasts. In the presence of CK2 inhibitors megakaryocytes matured to the pro-platelets bearing stage. Platelets were released through rupture, following cytoplasmic fragmentation and nuclear extrusion. Thrombocytopoiesis due to the use of CK2 inhibitors occurred both in suspension as well as with MEG-01 cells grown on a fibronectin matrix. Platelets obtained following these treatments were harvested and analyzed for function. Such platelets were identified as anucleated cells by flow cytometry and microscopy techniques using DAPI and PI staining. These platelets were found to undergo shape change in response to various agonists (human thrombin, TRAP, ADP, PMA, fibronectin). MEG-01-derived platelets were also found to stain positive with CD41a antibody (αIIbβ3) as determined by flow cytometry using a monoclonal antibody specific to the receptor. Following agonist activation, the platelets expose phosphatidylserine and P-Selectin. The specific use of PAC-1, an antibody that only recognizes the activated form of αIIbβ3, together with fibrinogen binding studies, demonstrated that the MEG-01-derived platelets can be activated. Fibronectin, RGDS and EDTA inhibited activation of MEG-01-derived platelets. Addition of human thrombin (5 U/ml) and fibrinogen to these platelets resulted in a stable clot. These findings suggest that CK2 is involved in MEG-01 differentiation, platelets production, and activation. Thus, by using a CK2 inhibitor we have successfully stopped the abnormal proliferation of a transformed cell line and reversed the path towards its normal function.

scholarly journals Optimization of pyrazolo[1,5-a]pyrimidines lead to the identification of a highly selective casein kinase 2 inhibitor

2020 ◽  
Author(s):  
Andreas Krämer ◽  
Christian Georg Kurz ◽  
Benedict-Tilman Berger ◽  
Ibrahim Ethem Celik ◽  
Stefan Knapp ◽  
...  
Keyword(s):  
Binding Mode ◽  
Casein Kinase ◽  
Casein Kinase 2 ◽  
Type I ◽  
Canonical Type ◽  
New Applications ◽  
Regulatory Functions ◽  
Ck2 Inhibitors ◽  
Ck2 Inhibitor

ABSTRACTCasein kinase 2 (CK2) is a constitutively expressed serine/threonine kinase that has a large diversity of cellular substrates. Thus, CK2 has been associated with a plethora of regulatory functions and dysregulation of CK2 has been linked to disease development in particular to cancer. The broad implications in disease pathology makes CK2 an attractive target. To date, the most advanced CK2 inhibitor is silmitasertib, which has been investigated in clinical trials for treatment of various cancers, albeit several off-targets for silmitasertib have been described. To ascertain the role of CK2 inhibition in cancer, other disease and normal physiology the development of a selective CK2 inhibitor would be highly desirable. In this study we explored the pyrazolo[1,5-a]pyrimidine hinge-binding moiety for the development of selective CK2 inhibitors. Optimization of this scaffold, which included macrocyclization, led to IC20 (31) a compound that displayed high in vitro potency for CK2 (KD = 12 nM) and exclusive selectivity for CK2. X-ray analysis revealed a canonical type-I binding mode for IC20. However, the polar carboxylic acid moiety that is shared by many CK2 inhibitors including silmitasertib was required for potency and reduced somewhat cellular activity. In summary, IC20 represents a highly selective and potent inhibitor of CK2, which can be used as a tool compound to study CK2 biology and potential new applications for the treatment of diseases.NotesThe authors declare no conflict of interest.TOC Figure / Graphical Abstract

scholarly journals TRPA1 block protects against loss of white matter function during ischaemia in the mouse optic nerve

2021 ◽  
Author(s):  
Grace Flower ◽  
Vincenzo Giacco ◽  
Angela Roxas ◽  
Nicola B Hamilton-Whitaker ◽  
Andra Braban
Keyword(s):  
White Matter ◽  
Optic Nerve ◽  
Action Potential ◽  
Neuronal Excitability ◽  
Glucose Deprivation ◽  
Compound Action Potential ◽  
Oxygen And Glucose Deprivation ◽  
Loss And Damage ◽  
Compound Action Potentials ◽  
Compound Action

Oligodendrocytes produce myelin which provides insulation to axons and speeds up neuronal transmission. In ischaemic conditions myelin is damaged, resulting to mental and physical disabilities. Therefore, it is important to understand how the functionality of oligodendrocytes and myelin is affected by ischaemia. Recent evidence suggests that oligodendrocyte damage during ischaemia is mediated by TRPA1, whose activation raises intracellular Ca2+ concentrations and damages compact myelin. Here, we show that TRPA1 is tonically active in oligodendrocytes and the optic nerve, as the specific TRPA1 antagonist, A-967079, decreases basal oligodendrocyte Ca2+ concentrations and increases the size of the compound action potential. Conversely, TRPA1 agonists reduce the size of the optic nerve compound action potential, and this effect is significantly reduced by the TRPA1 antagonist. These results indicate that glial TRPA1 regulates neuronal excitability in the white matter under physiological as well as pathological conditions. Importantly, we find that inhibition of TRPA1 prevents loss of compound action potentials during oxygen and glucose deprivation (OGD) and improves the recovery. TRPA1 block was effective when applied before, during or after OGD, indicating that the damage is occurring during ischaemia, but that therapeutic intervention is possible after the ischaemic insult. These results indicate that TRPA1 has an important role in the brain, and that its block may be effective in treating oligodendrocyte loss and damage in many white matter diseases.

Key Aspects of Assessing Effectiveness and Efficiency of Implementation of the Federal Clean Air Project on the Example of the Comprehensive Emission Reduction Action Plan in Nizhny Tagil

2020 ◽  
pp. 48-60
Author(s):  
SV Yarushin ◽  
DV Kuzmin ◽  
AA Shevchik ◽  
TM Tsepilova ◽  
VB Gurvich ◽  
...  
Keyword(s):  
Emission Reduction ◽  
Action Plan ◽  
Local Population ◽  
Ambient Air ◽  
Ambient Air Pollution ◽  
Mortality And Morbidity ◽  
Industrial Enterprises ◽  
Clean Air ◽  
Effectiveness And Efficiency ◽  
The Impact

Introduction: Key issues of assessing effectiveness and economic efficiency of implementing the Federal Clean Air Project by public health criteria are considered based on the example of the Comprehensive Emission Reduction Action Plan realized in the city of Nizhny Tagil, Sverdlovsk Region. Materials and methods: We elaborated method approaches and reviewed practical aspects of evaluating measures taken in 2018–2019 at key urban industrial enterprises accounting for 95 % of stationary source emissions. Results: Summary calculations of ambient air pollution and carcinogenic and non-carcinogenic inhalation health risks including residual risks, evaluation of the impact of air quality on urban mortality and morbidity rates, economic assessment of prevented morbidity and premature mortality cases have enabled us not only to estimate health effects but also to develop guidelines for development and implementation of actions aimed at enhancing effectiveness and efficiency of industrial emission reduction in terms of health promotion of the local population. Conclusions: We substantiate proposals for the necessity and sufficiency of taking remedial actions ensuring achievement of acceptable health risk levels as targets of the Comprehensive Emission Reduction Action Plan in Nizhny Tagil until 2024 and beyond.

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