Neuronal mdr-1 gene expression after experimental focal hypoxia: A new obstacle for neuroprotection?
Introduction
Stroke is the second leading cause of death and the first leading cause of serious long term disability [1], [2]. In normal conditions, neurons sustain neurotransmission and prevent that calcium and other ions of reaching toxic levels by using active transport to regulate the internal and external electrolyte milieu. The acute ischemia induces molecular time-dependent cascade events, initiated by decreased energy production and finally inducing neuronal death. The excitotoxicity due to over-stimulation of glutamate receptors, excessive intraneuronal accumulation of sodium, chloride, and calcium ions and the mitochondrial injury are the main players in the neuronal death [1], [3], [4].
Current medical strategies are focused on the blood flow restoration to recover normal oxygen supply as soon as possible after ischemia. Neuroprotective experimental strategies based on the interruption or slowdown of the mentioned cell-damage or death-cascade have been tested in clinical studies without success. Thus, at present the unique available treatment in humans is the tPA therapy related with the first stratagem, which is only recommended if it is initiated within 3 h after the onset of symptoms. Neuroprotection is not observed after this very short therapeutic window. This lack of effectiveness could be related with the biological consequences for brain, and particularly for neurons exposed longer time than 3 h to low or null oxygen level [5], [6], [7].
The ischemic penumbra surrounding the core is an area of reduced perfusion in which cells are still viable. These cells are subjected to various active processes in which several mechanisms are involved in their demise or their survival. Spontaneous reperfusion usually occurs after cerebral ischemia [8] and it may reverse the ischemic damage when occurring early enough (e.g., transient ischemic attacks) in concordance with the mentioned therapeutic window. However, it usually takes place at a much later time points, when most penumbral cells are already suffering the secondary damage [3]. These changes on behavior and phenotype of still alive brain parenchyma could be related with the lack of neuroprotective effects of the drugs.
In concert with this wide spectrum of biochemical modifications, and additionally to the brain–blood barrier (BBB) restrictive function, brain parenchyma cells can develop, upregulate or overexpress several selective and/or nonselective mechanisms that are able to prevent the intracellular drug accumulation [9], [10].
P-glycoprotein (P-gp), a well-known member of the ATP binding cassette transporters superfamily, is the product of the multiple drug resistance 1 (MDR-1) gene. P-gp is a transmembrane ATP-dependent efflux pump of cytotoxic compounds that confers refractory phenotype to expressive cells [11]. P-gp is normally expressed on the apical luminal surface of secretor cells in the small intestine, the colon and the proximal tubules of the kidney. In normal brain, P-glycoprotein is expressed in the endothelial cells of the BBB [12] supporting the concept of a protective role for MDR-1-type proteins in the removal of potentially toxic xeno- and endobiotics. We have previously demonstrated that, secondary to seizures or ischemic stress in rats, brain expression of P-gp is upregulated from normally non-expressive cells as astrocytes and neurons [13], [14], [15].
Several reports indicate that cobalt chloride (CoCl2) stabilizes or induce the accumulation of hypoxia-induced factor 1α (HIF-1α) [16], [17], a transcription factor that up-regulates several genes [18] including the mdr-1 gene which produces the P-gp protein [19]. CoCl2 has been widely used as a hypoxia-mimicking agent in both in vivo and in vitro studies [20], [21]. Cobalt is essential for human health because of its critical role in the synthesis of vitamin B12 [22], however, excess exposure of cobalt can lead to tissue and cellular toxicity [23].
Our hypothesis is that persistent hypoxic stress (as the one induced by CoCl2 intracerebral administration) could trigger several molecular and signaling pathways and upregulate P-gp expression. In accordance with another report on retinal degeneration by CoCl2 [23], we have shown that the intracortical injection of CoCl2 induces a localized brain damage similar to that found in other models of focal ischemia [24].
Since CoCl2 stabilizes active the form of HIF-1α [25], and mdr-1 gene is sensitive to HIF-1α, we investigated the pattern of P-gp expression in cerebral cortex after the injection of CoCl2 in the brain parenchyma.
Section snippets
Materials and methods
Mouse monoclonal anti-MDR1 (P-gp, clone C494, Signet), polyclonal rabbit anti GFAP (Glial Fibrillary Acidic Protein, Dako) and polyclonal rabbit anti NSE (Neuron Specific Enolase, Sigma) were used. Secondary biotinylated antibodies and streptavidin peroxidase complex (Extravidin) for immunohistochemistry studies were purchased from Sigma (USA). Fluorescent Fab2 anti-mouse or anti-rabbit were purchased from Immunotech (France). All other chemical substances were of analytical grade.
Results
The protocol for the CoCl2 injection used in this study resulted in a minimal physical disruption of the vasculature overlying part of the prefrontal cortex. CoCl2 induced a necrotic infarct restricted to the injection site and a penumbra area was observed in the surrounding tissue. The depth of the lesions was constant and controlled being the needle track easily observed both in the animals injected with saline solution or in those injected with CoCl2. The effect of the CoCl2 injection was
Discussion
Hypoxia response triggers the activation of the transcription factor HIF-1α leading to up-regulation of genes that are expressed in most cell types, such as those encoding glucose transporters, glycolytic enzymes, and vascular endothelial growth factor, as well as genes that are expressed in a cell type-specific manner, such as erythropoietin, inducible nitric oxide synthase, and insulin-like growth factor II [29].
The hypoxia responsive pathway is also specifically stimulated by exposure to CoCl
Acknowledgements
Supported by grants UBACYT M-072, CONICET PIP5034 (to A.B.) and PIP6063 (to A.J.R). A.J.R. and A.B. are researchers from CONICET (Argentina). We thank Mrs. Emerita Jorge Vilela de Bianchieri for her expert technical assistance with the EM studies, Dr. C. Gibson (Leicester University, UK) for the TTC protocol and Dr. Juana M. Pasquini and Dr. Laura Pasquini for the Olympus UV microscope.
References (50)
- et al.
Cerebral ischemia and trauma — different etiologies yet similar mechanisms: neuroprotective opportunities
Brain Res Rev
(2002) - et al.
Alteplase for acute stroke revisited: the first 10 years
Lancet Neurol
(2006) - et al.
Induction of hypoxia-inducible factor-1alpha overexpression by cobalt chloride enhances cellular resistance to photodynamic therapy
Cancer Lett
(2006) - et al.
Cobalt promotes angiogenesis via hypoxia-inducible factor and protects tubulointerstitium in the remnant kidney model
Lab Invest
(2005) - et al.
Characterization of hypoxia-inducible factor 1 and regulation of DNA binding activity by hypoxia
J Biol Chem
(1993) - et al.
Multiple biosynthetic pathways for vitamin B12: variations on a central theme
Vitam Horm
(2001) - et al.
A new model of retinal photoreceptor cell degeneration induced by a chemical hypoxia-mimicking agent, cobalt chloride
Brain Res
(2006) - et al.
Cobalt Inhibits the Interaction between hypoxia-inducible factor-a and von Hippel-Lindau protein by direct binding to hypoxia-inducible factor-a
J Biol Chem
(2003) - et al.
C. elegans EGL-9 and mammalian homologs define a family of dioxygenases that regulate HIF by prolyl hydroxylation
Cell
(2001) - et al.
Blood–brain barrier active efflux transporters: ATPbinding cassette gene family
NeuroRx
(2005)
P-glycoprotein protects leukemia cells agonist caspase-dependent but not caspase-independent cell death
Blood
Redundancy of biological regulation as the basis of emergence of multidrug resistance
Int Rev Cytol
Delivery of therapeutic agents to the central nervous system: the problems and the possibilities
Pharmacol Ther
Biochemical, cellular and molecular mechanisms in the evolution of secondary damage after severe traumatic brain injury in infants and children: lessons learned from the bedside
Pediatr Crit Care
Release of caspase-9 from mitochondria during neuronal apoptosis and cerebral ischemia
Proc Natl Acad Sci U S A
A systems approach to immediate evaluation and management of hyperacute stroke: experience at eight centers and implications for community practice and patient care
Stroke
Treatment of acute ischemic stroke
N Engl J Med
Stroke Council of the American Stroke Association. Guidelines for the early management of patients with ischemic stroke: A scientific statement from the Stroke Council of the American Stroke Association
Stroke
Thrombolytic reversal of acute human cerebral ischemic injury shown by diffusion/perfusion magnetic resonance imaging
Ann Neurol
Drug transporters in the central nervous system: brain barriers and brain parenchyma considerations
Pharmacol Rev
A novel zidovudine uptake system in microglia
J Pharmacol Exp Ther
Multidrug resistance in cancer: role of ATP-dependent transporters
Nat Rev Cancer
Expression of the multidrug resistance gene product (P-glycoprotein) in human normal and tumor tissues
J Histochem Cytochem
Transient Expression of MDR-1/P-Glycoprotein in a Model of Partial Cortical Devascularization
Cell Mol Neurobiol
Neuronal and glial expression of the multidrug resistance gene product in an experimental epilepsy model
Cell Mol Neurobiol
Cited by (55)
Combination Organelle Mitochondrial Endoplasmic Reticulum Therapy (COMET) for Multidrug Resistant Breast Cancer
2023, Journal of Controlled ReleaseImportance of selected ABCB1 SNPs for the level of severity of depressive symptoms and effectiveness of recurrent depressive disorder therapy
2023, GeneCitation Excerpt :In the central nervous system, P-gp is normally expressed in the blood–brain barrier (BBB) (Spudich et al., 2006). Neuronal expression of ATP-binding cassette, sub-family B, member 1 (ABCB1) has been demonstrated in some diseases, e.g. after brain ischemia (Lazarowski et al., 2007). The level of mRNA and also function of P-gp are strictly connected with the polymorphic nature of the ABCB1 gene.
Lithium enhances post-stroke blood-brain barrier integrity, activates the MAPK/ERK1/2 pathway and alters immune cell migration in mice
2020, NeuropharmacologyCitation Excerpt :A study in P-gp knockout mice suggests that the reduction of P-gp expression is neuroprotective (Murozono et al., 2009). On the other hand, P-gp has been shown to protect cells from intracellular accumulation of toxic components and was found to be elevated in hypoxia-induced apoptosis in cells (Johnstone et al., 1999; Lazarowski et al., 2007; Robinson et al., 1997). Moreover, Kraya et al. suggest that P-gp activity positively affects tight junctional protein expression and BBB resistance (Kraya et al., 2016).
Dysfunction of ABC transporters at the blood-brain barrier: Role in neurological disorders
2020, Pharmacology and TherapeuticsCitation Excerpt :Also, this increase in P-gp expression and activity was reflected in a significant decrease of the intracellular accumulation of phenobarbital to only 62.2% of control cells (Xiao-Dong et al., 2008). These results reinforce that tissue stress (e.g., hypoxia) can lead to P-gp upregulation (Felix & Barrand, 2002; Lazarowski et al., 2007; Robertson et al., 2009; Xiao-Dong et al., 2008). In addition, ElAli et al. reported that APOE regulates post-ischemic abundance of P-gp and MRP1 through a JNK1/JNK2 dependent mechanism, which can theoretically modulate the accumulation of brain therapeutics in the ischemic brain (ElAli & Hermann, 2010).
HMGB1 promoted P-glycoprotein at the blood-brain barrier in MCAO rats via TLR4/NF-κB signaling pathway
2020, European Journal of PharmacologyCitation Excerpt :60 min before the occlusion, EP (50 mg/kg), TAK-242 (1.5 mg/kg) and PDTC (100 mg/kg) were administered intraperitoneally. 3 h after MCAO, brain infarct volume was determined by 2,3,5-Triphenyl-2H-tetrazolium chloride (TTC) staining (Lazarowski et al., 2007). After intraperitoneal injection of pentobarbital (100 mg/kg), the brain was quickly removed and the 2 mm slices were cut from the frontal pole to the occipital pole and stained in 2% TTC at 37 °C for 30 min.
Polyamine analogue QMA attenuated ischemic injury in MCAO rats via ERK and Akt activated Nrf2/HO-1 signaling pathway
2019, European Journal of Pharmacology
- 1
Present address: Department of Cell Biology and Neuroscience, University of California Riverside, USA.