The Proteomic Analysis of Primary Cortical Astrocyte Cell Culture after Morphine Administration

Suder, Piotr; Bodzoń‐Kułakowska, Anna; Mak, Paweł; Bierczyńska-Krzysik, Anna; Daszykowski, M.; Walczak, Beata; Lubec, Gert; Kotlińska, Jolanta; Silberring, Jerzy

doi:10.1021/pr900443r

Cited by 28 publications

(22 citation statements)

References 70 publications

(125 reference statements)

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“…It has been shown that acute morphine stimulation can induce calcium signaling El-Hage et al, 2005) and ERK phosphorylation (Belcheva et al, 2003), while prolonged exposure affected proliferation (Stiene-Martin et al, 2001) and proteomic profile (Suder et al, 2009) in primary astrocytes. The absence of direct transcriptional effects of morphine in astrocytes in the current study may be explained by the low level of l opioid receptors in our cultures, which is consistent with previous reports (Ruzicka et al, 1995;Stiene-Martin et al, 1998).…”

Section: Discussion the Gc-dependent Component Of Morphineinduced Sigmentioning

confidence: 99%

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Astrocytes are a neural target of morphine action via glucocorticoid receptor‐dependent signaling

Ślęzak

Korostyński

Gieryk

et al. 2013

Glia

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Chronic opioid use leads to the structural reorganization of neuronal networks, involving genetic reprogramming in neurons and glial cells. Our previous in vivo studies have revealed that a significant fraction of the morphine-induced alterations to the striatal transcriptome included glucocorticoid (GC) receptor (GR)-dependent genes. Additional analyses suggested glial cells to be the locus of these changes. In the current study, we aimed to differentiate the direct transcriptional effects of morphine and a GR agonist on primary striatal neurons and astrocytes. Whole-genome transcriptional profiling revealed that while morphine had no significant effect on gene expression in both cell types, dexamethasone significantly altered the transcriptional profile in astrocytes but not neurons. We obtained a complete dataset of genes undergoing the regulation, which includes genes related to glucose metabolism (Pdk4), circadian activity (Per1) and cell differentiation (Sox2). There was also an overlap between morphine-induced transcripts in striatum and GR-dependent transcripts in cultured astrocytes. We further analyzed the regulation of expression of one gene belonging to both groups, serum and GC regulated kinase 1 (Sgk1). We identified two transcriptional variants of Sgk1 that displayed selective GR-dependent upregulation in cultured astrocytes but not neurons. Moreover, these variants were the only two that were found to be upregulated in vivo by morphine in a GR-dependent fashion. Our data suggest that the morphine-induced, GR-dependent component of transcriptome alterations in the striatum is confined to astrocytes. Identification of this mechanism opens new directions for research on the role of astrocytes in the central effects of opioids. V V C 2013 Wiley Periodicals, Inc.

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Section: Discussion the Gc-dependent Component Of Morphineinduced Sigmentioning

confidence: 99%

“…This concentration has been used previously to estimate the proteome changes after chronic morphine treatment (Suder et al, 2009). The dexamethasone concentration was based on pilot studies in which a dose of 10 nM was the minimum that effectively induced the expression of selected GR-dependent genes.…”

Section: Materials and Methods Cell Culturementioning

confidence: 99%

Astrocytes are a neural target of morphine action via glucocorticoid receptor‐dependent signaling

Ślęzak

Korostyński

Gieryk

et al. 2013

Glia

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“…In addition, a number of studies also observed consistent and significant morphine-regulated alterations in the expression of chaperone proteins, specifically heat shock proteins including Hsp60 and Hsp70 (Prokai et al, 2005;Shui et al, 2007;Yang et al, 2007;Suder et al, 2009;Bu et al, 2012). Heat shock proteins such as HSP70 and HSP90 often serve as molecular chaperones, and both iTRAQ þ MS/MS analysis and western Blotting analyses have confirmed the presence of significantly elevated levels of HSP70 in the striatal PSD fraction following the induction of morphine dependence.…”

Section: Discussionmentioning

confidence: 95%

“…Moreover, utilizing an unbiased methodology also generates data with respect to the enhanced or reduced expression of proteins in response to the physiological perturbation applied. Two-dimensional electrophoresis (2-DE) represents the most common approach utilized to assess morphine-regulated alterations in protein expression (Table 1) (Kim et al, , 2005Li et al, 2006Li et al, , 2009Shui et al, 2007;Yang et al, 2007;Suder et al, 2009;Tan et al, 2010;Lin et al, 2011;Bu et al, 2012;Song et al, 2012;Wei et al, 2013). One of the earliest studies that utilized this approach studied alterations in the morphinome resulting from behavioral sensitization induced as a consequence of 14 days of intermittent exposure to morphine (Li et al, 2006) (Morphinome refers to a set of proteins at a brain region affected by morphine treatment.)…”

Section: Neuroproteomics Explorations Of Morphinomementioning

confidence: 99%

An Integrated Quantitative Proteomics and Systems Biology Approach to Explore Synaptic Protein Profile Changes During Morphine Exposure

Stockton

Devi

2013

Neuropsychopharmacol

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Morphine is a classic analgesic for the treatment of chronic pain. However, its repeated use is known to produce tolerance, physical dependence, and addiction; these properties limit its long-term therapeutic use and this has led to a quest for therapeutics without these unwanted side effects. Understanding the molecular changes in response to long-term use of morphine is likely to aid in the development of novel therapeutics for the treatment of pain. Studies examining the effects of chronic morphine administration have reported alterations in gene expression, synapse morphology, and synaptic transmission implying changes in synaptic protein profile. To fully understand the changes in protein profiles, proteomic techniques have been used. Studies using two-dimensional gel electrophoresis of various brain regions combined with mass spectrometry have found alterations in the levels of a number of proteins. However, neither the changes in brain regions relevant to morphine effects nor changes in the abundance of synaptic proteins have been clearly delineated. Recent studies employing subcellular fractionation to isolate the striatal synapse, combined with quantitative proteomics and graph theoryinspired network analyses, have begun to quantify morphine-regulated changes in synaptic proteins and facilitate the generation of networks that could serve as targets for the development of novel therapeutics for the treatment of chronic pain. Thus, an integrated quantitative proteomics and systems biology approach can be useful to identify novel targets for the treatment of pain and other disorders of the brain.

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“…Proteomic analysis has been used to elucidate both the in vitro (18,19) and in vivo (20 -26) changes in the protein profiles of synapses that exhibit drug-induced alterations in response to opiate exposure. For example, the use of subcellular fractionation, which allows for the separation and generation of protein fractions selectively enriched in either pre-or postsynaptic proteins (25,27), in combination with proteomic analysis has revealed that an acute escalating dose of morphine administration produces significant alterations in the postsynaptic density (PSD) 1 associated protein network in the hippocampus.…”

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confidence: 99%

Morphine Regulated Synaptic Networks Revealed by Integrated Proteomics and Network Analysis

Stockton¹,

Gomes²,

Liu³

et al. 2015

Molecular & Cellular Proteomics

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Despite its efficacy, the use of morphine for the treatment of chronic pain remains limited because of the rapid development of tolerance, dependence and ultimately addiction. These undesired effects are thought to be because of alterations in synaptic transmission and neuroplasticity within the reward circuitry including the striatum. In this study we used subcellular fractionation and quantitative proteomics combined with computational approaches to investigate the morphine-induced protein profile changes at the striatal postsynaptic density. Over 2,600 proteins were identified by mass spectrometry analysis of subcellular fractions enriched in postsynaptic density associated proteins from saline or morphine-treated striata. Among these, the levels of 34 proteins were differentially altered in response to morphine. These include proteins involved in G-protein coupled receptor signaling, regulation of transcription and translation, chaperones, and protein degradation pathways. The altered expression levels of several of these proteins was validated by Western blotting analysis. Using Genes2Fans software suite we connected the differentially expressed proteins with proteins identified within the known background protein-protein interaction network. This led to the generation of a network consisting of 116 proteins with 40 significant intermediates. To validate this, we confirmed the presence of three proteins predicted to be significant intermediates: caspase-3, receptor-interacting serine/threonine protein kinase 3 and NEDD4 (an E3-ubiquitin ligase identified as a neural precursor cell expressed developmentally down-regulated protein 4). Because this morphine-regulated network predicted alterations in proteasomal degradation, we examined the global ubiquitination state of postsynaptic density proteins and found it to be substantially altered. Together, these findings suggest a role for protein degradation and for the ubiquitin/proteasomal system in the etiology of opiate dependence and addiction. Morphine and other opiates are the drugs of choice for the treatment of both severe and chronic pain. However, the utility of these compounds in the clinical setting is limited because of the rapid development of tolerance, physical dependence and addiction. The underlying cellular and molecular alterations through which chronic opiate exposure results in the persistent behavioral phenomenon of addiction remain poorly understood. However, there is evidence suggesting that the molecular composition of synapses within the reward circuitry of the central nervous system, particularly in the striatum, may be significantly altered (1). Moreover, given the critical involvement of the striatum in translating emotional or rewarding stimuli into motivated behaviors, alterations in this region induced by morphine and other abused drugs could contribute significantly to the pathophysiological responses at the heart of addiction (2). Although the debate surrounding the molecular and cellular mechanisms by which repeated drug adminis...

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The Proteomic Analysis of Primary Cortical Astrocyte Cell Culture after Morphine Administration

Cited by 28 publications

References 70 publications

Astrocytes are a neural target of morphine action via glucocorticoid receptor‐dependent signaling

Astrocytes are a neural target of morphine action via glucocorticoid receptor‐dependent signaling

An Integrated Quantitative Proteomics and Systems Biology Approach to Explore Synaptic Protein Profile Changes During Morphine Exposure

Morphine Regulated Synaptic Networks Revealed by Integrated Proteomics and Network Analysis

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