Publications

Transfection with GLS2 Glutaminase (GAB) Sensitizes Human Glioblastoma Cell Lines to Oxidative Stress by a Common Mechanism Involving Suppression of the PI3K/AKT Pathway

Ewelina Majewska 1, JavierMárquez 2 , Jan Albrecht 1 and Monika Szeliga 1,*
1 Department of Neurotoxicology, Mossakowski Medical Research Centre, Polish Academy of Sciences, 5 Pawinskiego Street, 02-106 Warsaw, Poland; This email address is being protected from spambots. You need JavaScript enabled to view it. (E.M.); This email address is being protected from spambots. You need JavaScript enabled to view it. (J.A.)
2 Canceromics Laboratory, Department of Molecular Biology and Biochemistry, Faculty of Sciences, Campus de Teatinos, Instituto de Investigación Biomédica de Málaga (IBIMA), University of Málaga, 29071 Málaga, Spain; This email address is being protected from spambots. You need JavaScript enabled to view it.
* Correspondence: This email address is being protected from spambots. You need JavaScript enabled to view it.; Tel.: +48-22-60-86-416
Received: 27 December 2018; Accepted: 17 January 2019; Published: 19 January 2019

Abstract: GLS-encoded glutaminase promotes tumorigenesis, while GLS2-encoded glutaminase displays tumor-suppressive properties. In glioblastoma (GBM), the most aggressive brain tumor, GLS is highly expressed and in most cases GLS2 is silenced. Previously, it was shown that transfection with a sequence encoding GAB, the main GLS2 isoform, decreased the survival, growth, and ability to migrate of human GBM cells T98G and increased their sensitivity towards an alkylating agent temozolomide (TMZ) and oxidative stress compared to the controls, by a not well-defined mechanism. In this study we report that GAB transfection inhibits growth and increases susceptibility towards TMZ and H2O2-mediated oxidative stress of two other GBM cell lines, U87MG and LN229. We also show that in GAB-transfected cells treated with H2O2, the PI3K/AKT pathway is less induced compared to the pcDNA-transfected counterparts and that pretreatment with PDGF-BB, an activator of AKT, protects GAB-transfected cells from death caused by the H2O2 treatment. In conclusion, our results show that (i) GAB suppresses the malignant phenotype of the GBM cells of different tumorigenic potentials and genetic backgrounds and (ii) the GAB-mediated increase of sensitivity to oxidative stress is causally related to the inhibition of the PI3K/AKT pathway. The upregulation of the GLS2 expression and the inhibition of the PI3K/AKT pathway may become a novel combined therapeutic strategy for anti-glioma preclinical investigations.
Keywords: GLS2 glutaminase; human glioblastoma; PI3K/AKT signaling pathway; oxidative stress.

Results Probl Cell Differ. 2018;66:207-230. doi: 10.1007/978-3-319-93485-3_9.

Bioengineering of the Human Neural Stem Cell Niche: A Regulatory Environment for Cell Fate and Potential Target for Neurotoxicity.
 
Buzanska L 1, Zychowicz M 2, Kinsner-Ovaskainen A 3.
 
Author information:

1 Stem Cell Bioengineering Unit, Mossakowski Medical Research Centre Polish Academy of Sciences, Warsaw, Poland. This email address is being protected from spambots. You need JavaScript enabled to view it..

2 Stem Cell Bioengineering Unit, Mossakowski Medical Research Centre Polish Academy of Sciences, Warsaw, Poland.

3 European Commission, Joint Research Centre, Directorate for Health Consumers and Reference Materials, Ispra, Italy.

ABSTRACT:

Human neural stem/progenitor cells of the developing and adult organisms are surrounded by the microenvironment, so-called neurogenic niche. The developmental processes of stem cells, such as survival, proliferation, differentiation, and fate decisions, are controlled by the mutual interactions between cells and the niche components. Such interactions are tissue specific and determined by the biochemical and biophysical properties of the niche constituencies and the presence of other cell types. This dynamic approach of the stem cell niche, when translated into in vitro settings, requires building up "biomimetic" microenvironments resembling natural conditions, where the stem/progenitor cell is provided with diverse extracellular signals exerted by soluble and structural cues, mimicking those found in vivo. The neural stem cell niche is characterized by a unique composition of soluble components including neurotransmitters and trophic factors as well as insoluble extracellular matrix proteins and proteoglycans. Biotechnological innovations provide tools such as a new generation of tunable biomaterials capable of releasing specific signals in a spatially and temporally controlled manner, thus creating in vitro nature-like conditions and, when combined with stem cell-derived tissue specific progenitors, producing differentiated neuronal tissue structures. In addition, substantial progress has been made on the protocols to obtain stem cell-derived cell aggregates such as neurospheres and self-assembled organoids.In this chapter, we have assessed the application of bioengineered human neural stem cell microenvironments to produce in vitro models of different levels of biological complexity for the efficient control of stem cell fate. Examples of biomaterial-supported two-dimensional and three-dimensional (2D and 3D) complex culture systems that provide artificial neural stem cell niches are discussed in the context of their application for basic research and neurotoxicity testing.
 
 
KEYWORDS:

Bioengineering; Neural stem cells; Neurotoxicity; Stem cell niche

Persistent Overexposure to N-Methyl-D-Aspartate (NMDA) Calcium-Dependently Downregulates Glutamine Synthetase, Aquaporin 4, and Kir4.1 Channel in Mouse Cortical Astrocytes.

Neurotox Res. 2019 Jan;35(1):271-280. doi: 10.1007/s12640-018-9958-3. Epub 2018 Sep 15.

Skowrońska K1, Obara-Michlewska M1, Czarnecka A1, Dąbrowska K1, Zielińska M1, Albrecht J2.

Author information:

1     Department of Neurotoxicology, Mossakowski Medical Research Centre, Polish Academy of Sciences, Pawińskiego St. 5, 02-106, Warsaw, Poland.

2      Department of Neurotoxicology, Mossakowski Medical Research Centre, Polish Academy of Sciences, Pawińskiego St. 5, 02-106, Warsaw, Poland. This email address is being protected from spambots. You need JavaScript enabled to view it..

Abstract

Astrocytes express N-methyl-D-aspartate (NMDA) receptor (NMDAR) but its functions in these cells are not well defined. This study shows that the sustained exposure (8-72 h) of mouse astrocytes to NMDA decreases the expression of the functional astroglia-specific proteins, glutamine synthetase (GS), and the water channel protein aquaporin-4 (AQP4) and also reduces GS activity. Similar to rat astrocytes (Obara-Michlewska et al. Neurochem Int 88:20-25, 2015), the exposure of mouse astrocytes to NMDA also decreased the expression of the inward rectifying potassium channel Kir4.1. NMDA failed to elicit the effects in those cells incubated in the absence of Ca2+ and in those in which the GluN1 subunit of the NMDAR was silenced with GluN1 siRNA. The downregulation of GS, AQP4, and Kir4.1 observed in vitro may reflect NMDAR-mediated alterations of astrocytic functions noted in central nervous system pathologies associated with increased glutamate (Glu) release and excitotoxic tissue damage.

KEYWORDS:

Aquaporin 4; Astrocytes; Excitotoxicity; GluN1; Glutamine synthetase; Kir4.1; NMDA receptor

Cerebrovascular reactivity and cerebral perfusion of rats with acute liver failure: role of L-glutamine and asymmetric dimethylarginine in L-arginine-induced response.

J Neurochem. 2018 Dec;147(5):692-704. doi: 10.1111/jnc.14578. Epub 2018 Nov 6.

 Czarnecka A 1, Aleksandrowicz M 2, Jasiński K 3, Jaźwiec R 4, Kalita K 3, Hilgier W 1, Zielińska M 1.

Author information:

1   Department of Neurotoxicology, Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland.

2   Department of Neurosurgery, Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland.

3   Department of Magnetic Resonance Imaging, Institute of Nuclear Physics, Polish Academy of Sciences, Kraków, Poland.

4    Mass Spectrometry Laboratory, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland.

Abstract:

Cerebral blood flow (CBF) is impaired in acute liver failure (ALF), however, the complexity of the underlying mechanisms has often led to inconclusive interpretations. Regulation of CBF depends at least partially on variations in the local brain L-arginine concentration and/or its metabolic rate. In ALF, other factors, like an increased concentration of asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase inhibitor and elevated level of L-glutamine, may contribute to CBF alteration. This study demonstrated strong differences in the reactivity of the middle cerebral arteries and their response to extravascular L-arginine application between vessels isolated from rats with thioacetamide (TAA)-induced ALF and control animals. Our results also showed the decrease in the cerebral perfusion in TAA rats measured by arterial spin labeling perfusion magnetic resonance. Subsequently, we aimed to investigate the importance of balance between the concentration of ADMA and L-arginine in the CBF regulation. In vivo, intraperitoneal L-arginine administration in TAA rats corrected: (i) decrease in cerebral perfusion, (ii) decrease in brain extracellular L-arginine/ADMA ratio and (iii) increase in brain L-glutamine concentration. Our study implicates that impaired vascular tone of cerebral arteries is most likely associated with exposure to high ADMA and L-glutamine levels resulting in limited availability of L-arginine and might be responsible for reduced cerebral perfusion observed in ALF.

KEYWORDS:

Acute liver failure; MRI-ASL; asymmetric dimethylarginine; cerebral blood flow; isolated middle cerebral artery

Inhibition of cyclin-dependent kinase 5 affects early neuroinflammatory signalling in murine model of amyloid beta toxicity.

Wilkaniec A1, Gąssowska-Dobrowolska M1, Strawski M2, Adamczyk A1, Czapski GA3.

Author information:

1 Department of Cellular Signalling, Mossakowski Medical Research Centre Polish Academy of Sciences, Pawińskiego 5, 02-106, Warsaw, Poland.

2 Laboratory of Electrochemistry, Faculty of Chemistry, University of Warsaw, Pasteura 1, 02-093, Warsaw, Poland.

3 Department of Cellular Signalling, Mossakowski Medical Research Centre Polish Academy of Sciences, Pawińskiego 5, 02-106, Warsaw, Poland. This email address is being protected from spambots. You need JavaScript enabled to view it..

 

ABSTRACT:

BACKGROUND:

Cyclin-dependent kinase 5 (Cdk5) belongs to the family of proline-directed serine/threonine kinases and plays a critical role in neuronal differentiation, migration, synaptogenesis, plasticity, neurotransmission and apoptosis. The deregulation of Cdk5 activity was observed in post mortem analysis of brain tissue of Alzheimer's disease (AD) patients, suggesting the involvement of Cdk5 in the pathomechanism of this neurodegenerative disease. However, our recent study demonstrated the important function of Cdk5 in regulating inflammatory reaction.

 

METHODS:

Since the role of Cdk5 in regulation of inflammatory signalling in AD is unknown, we investigated the involvement of Cdk5 in neuroinflammation induced by single intracerebroventricular (icv) injection of amyloid beta protein (Aβ) oligomers in mouse. The brain tissue was analysed up to 35 days post injection. Roscovitine (intraperitoneal administration) was used as a potent Cdk5 inhibitor. The experiments were also performed on human neuroblastoma SH-SY5Y as well as mouse BV2 cell lines treated with exogenous oligomeric Aβ.

 

RESULTS:

Our results demonstrated that single injection of Aβ oligomers induces long-lasting activation of microglia and astrocytes in the hippocampus. We observed also profound, early inflammatory response in the mice hippocampus, leading to the significant elevation of pro-inflammatory cytokines expression (e.g. TNF-α, IL-1β, IL-6). Moreover, Aβ oligomers elevated the formation of truncated protein p25 in mouse hippocampus and induced overactivation of Cdk5 in neuronal cells. Importantly, administration of roscovitine reduced the inflammatory processes evoked by Aβ in the hippocampus, leading to the significant decrease of cytokines level.

 

CONCLUSIONS:

These studies clearly show the involvement of Cdk5 in modulation of brain inflammatory response induced by Aβ and may indicate this kinase as a novel target for pharmacological intervention in AD.

 

KEYWORDS:

Alzheimer’s disease; Amyloid beta; Cdk5; Cytokines; Gene expression; Neuroinflammation

Extracellular Alpha-Synuclein Oligomers Induce Parkin S-Nitrosylation: Relevance to Sporadic Parkinson's Disease Etiopathology.

Mol Neurobiol. 2018 Apr 21. doi: 10.1007/s12035-018-1082-0. [Epub ahead of print]

Wilkaniec A1, Lenkiewicz AM1, Czapski GA1, Jęśko HM1, Hilgier W2, Brodzik R3, Gąssowska-Dobrowolska M1, Culmsee C4, Adamczyk A5.

Author information:

1 Department of Cellular Signalling, Mossakowski Medical Research Centre, Polish Academy of Sciences, Pawińskiego 5 Street, 02-106, Warsaw, Poland.

2 Department of Neurotoxicology, Mossakowski Medical Research Centre, Polish Academy of Sciences, Pawińskiego 5 Street, 02-106, Warsaw, Poland.

3 BLIRT S.A., Trzy Lipy 3/1.38, 80-172, Gdańsk, Poland.

4 Institute of Pharmacology and Clinical Pharmacy, University of Marburg, 35043, Marburg, Germany.

5 Department of Cellular Signalling, Mossakowski Medical Research Centre, Polish Academy of Sciences, Pawińskiego 5 Street, 02-106, Warsaw, Poland. This email address is being protected from spambots. You need JavaScript enabled to view it..

 

ABSTRACT:

α-Synuclein (ASN) and parkin, a multifunctional E3 ubiquitin ligase, are two proteins that are associated with the pathophysiology of Parkinson's disease (PD). Excessive release of ASN, its oligomerization, aggregation, and deposition in the cytoplasm contribute to neuronal injury and cell death through oxidative-nitrosative stress induction, mitochondrial impairment, and synaptic dysfunction. In contrast, overexpression of parkin provides protection against cellular stresses and prevents dopaminergic neural cell loss in several animal models of PD. However, the influence of ASN on the function of parkin is largely unknown. Therefore, the aim of this study was to investigate the effect of extracellular ASN oligomers on parkin expression, S-nitrosylation, as well as its activity. For these investigations, we used rat pheochromocytoma (PC12) cell line treated with exogenous oligomeric ASN as well as PC12 cells with parkin overexpression and parkin knock-down. The experiments were performed using spectrophotometric, spectrofluorometric, and immunochemical methods. We found that exogenous ASN oligomers induce oxidative/nitrosative stress leading to parkin S-nitrosylation. Moreover, this posttranslational modification induced the elevation of parkin autoubiquitination and degradation of the protein. The decreased parkin levels resulted in significant cell death, whereas parkin overexpression protected against toxicity induced by extracellular ASN oligomers. We conclude that lowering parkin levels by extracellular ASN may significantly contribute to the propagation of neurodegeneration in PD pathology through accumulation of defective proteins as a consequence of parkin degradation.

KEYWORDS:

Alpha-synuclein; Parkin; Parkinson’s disease; S-nitrosylation

Neuroprotective Potential and Paracrine Activity of Stromal Vs. Culture-Expanded hMSC Derived from Wharton Jelly under Co-Cultured with Hippocampal Organotypic Slices.

Mol Neurobiol. 2018 Jul;55(7):6021-6036. doi: 10.1007/s12035-017-0802-1. Epub 2017 Nov 13.

Dabrowska S 1, Sypecka J 1, Jablonska A 1, Strojek L 1, Wielgos M 2, Domanska-Janik K 1,
Sarnowska A 3.

1 Mossakowski Medical Research Centre, Polish Academy of Sciences, 5 Pawinskiego Street, Warsaw, Poland.

2 1st Department of Obstetrics and Gynecology, Medical University of Warsaw, Warsaw, Poland.

3 Mossakowski Medical Research Centre, Polish Academy of Sciences, 5 Pawinskiego Street, Warsaw, Poland. This email address is being protected from spambots. You need JavaScript enabled to view it..

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Intra-arterial delivery of cell therapies for stroke.

Stroke. 2018 May;49(5):1075-1082. doi: 10.1161/STROKEAHA.117.018288. Epub 2018 Apr 18.

Guzman R 1, Janowski M 2,3,4,5, Walczak P 6,3,7.

1 From the Department of Neurosurgery and Biomedicine, University Hospital Basel, University of Basel, Switzerland (R.G.).

2 Russell H. Morgan Department of Radiology and Radiological Science, the Johns Hopkins University School of Medicine, Baltimore, MD (M.J., P.W.).

3 Cellular Imaging Section and Vascular Biology Program, Institute for Cell Engineering, the Johns Hopkins University School of Medicine, Baltimore, MD (M.J., P.W.).

4 NeuroRepair Department, Mossakowski Medical Research Centre, Polish Academy of Sciences (M.J.).

5 Department of Neurosurgery, Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland (M.J.).

6 Russell H. Morgan Department of Radiology and Radiological Science, the Johns Hopkins University School of Medicine, Baltimore, MD (M.J., P.W.) This email address is being protected from spambots. You need JavaScript enabled to view it..

7 Department of Neurology and Neurosurgery, Faculty of Medical Sciences, University of Warmia and Mazury, Olsztyn, Poland (P.W.).

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Hydrogel-based scaffolds to support intrathecal stem cell transplantation as a gateway to the spinal cord: clinical needs, biomaterials, and imaging technologies.

NPJ Regen Med. 2018 Apr 4;3:8. doi: 10.1038/s41536-018-0046-3. eCollection 2018.

Oliveira JM1,2,3, Carvalho L1,2, Silva-Correia J1,2, Vieira S1,2, Majchrzak M4, Lukomska B4, Stanaszek L4, Strymecka P4, Malysz-Cymborska I5, Golubczyk D5, Kalkowski L5, Reis RL1,2,3, Janowski M4,6,7, Walczak P5,6,7.

1 3B´s Research Group - Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence, Tissue Engineering and Regenerative Medicine, AvePark, Zona Industrial da Gandra, 4805-017 Barco, Guimarães Portugal.

2 2ICVS/3B's - PT Government Associate Laboratory, Braga, Portugal.

3 3The Discoveries Centre for Regenerative and Precision Medicine, Headquarters at University of Minho, Avepark, 4805-017 Barco, Guimarães Portugal.

4 4NeuroRepair Department, Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland.

5 5Department of Neurology and Neurosurgery, School of Medicine, Collegium Medicum, University of Warmia and Mazury, Olsztyn, Poland.

6 6Russel H, Morgan Department of Radiology and Radiological Science, Johns Hopkins University, Baltimore, MD USA.

7 7Vascular Biology Program, Institute for Cell Engineering, Johns Hopkins University, Baltimore, MD USA.

Hydrogel-based scaffolds to support intrathecal stem cell transplantation as a gateway to the spinal cord: clinical needs, biomaterials, and imaging technologies.

ABSTRACT

The prospects for cell replacement in spinal cord diseases are impeded by inefficient stem cell delivery. The deep location of the spinal cord and complex surgical access, as well as densely packed vital structures, question the feasibility of the widespread use of multiple spinal cord punctures to inject stem cells. Disorders characterized by disseminated pathology are particularly appealing for the distribution of cells globally throughout the spinal cord in a minimally invasive fashion. The intrathecal space, with access to a relatively large surface area along the spinal cord, is an attractive route for global stem cell delivery, and, indeed, is highly promising, but the success of this approach relies on the ability of cells (1) to survive in the cerebrospinal fluid (CSF), (2) to adhere to the spinal cord surface, and (3) to migrate, ultimately, into the parenchyma. Intrathecal infusion of cell suspension, however, has been insufficient and we postulate that embedding transplanted cells within hydrogel scaffolds will facilitate reaching these goals. In this review, we focus on practical considerations that render the intrathecal approach clinically viable, and then discuss the characteristics of various biomaterials that are suitable to serve as scaffolds. We also propose strategies to modulate the local microenvironment with nanoparticle carriers to improve the functionality of cellular grafts. Finally, we provide an overview of imaging modalities for in vivo monitoring and characterization of biomaterials and stem cells. This comprehensive review should serve as a guide for those planning preclinical and clinical studies on intrathecal stem cell transplantation.