Acute and Chronic Mu Opioids Differentially Regulate Thrombospondins 1 and 2 Isoforms in Astrocytes

Abstract

Chronic opioids induce synaptic plasticity, a major neuronal adaptation. Astrocyte activation in synaptogenesis may play a critical role in opioid tolerance, withdrawal, and dependence. Thrombospondins 1 and 2 (TSP1/2) are astrocyte-secreted matricellular glycoproteins that promote neurite outgrowth as well as dendritic spine and synapse formation, all of which are inhibited by chronic μ opioids. In prior studies, we discovered that the mechanism of TSP1 regulation by μ opioids in astrocytes involves crosstalk between three different classes of receptors, μ opioid receptor, EGFR and TGFβR. Moreover, TGFβ1 stimulated TSP1 expression via EGFR and ERK/MAPK activation, indicating that EGFR is a signaling hub for opioid and TGFβ1 actions. Using various selective antagonists, and inhibitors, here we compared the mechanisms of chronic opioid regulation of TSP1/2 isoform expression in vivo and in immortalized rat cortical astrocytes. TSP1/2 release from astrocytes was also monitored. Acute and chronic μ opioids, morphine, and the prototypic μ ligand, DAMGO, modulated TSP2 protein levels. TSP2 but not TSP1 protein content was up-regulated by acute (3 h) morphine or DAMGO by an ERK/MAPK dependent mechanism. Paradoxically, TSP2 protein levels were altered neither by TGFβ1 nor by astrocytic neurotrophic factors, EGF, CNTF, and BMP4. TSP1/2 immunofluorescence was increased in astrocytes subjected to scratch-wounding, suggesting TSPs may be useful markers for the "reactive" state of these cells and potentially for different types of injury. Previously, we determined that chronic morphine attenuated both neurite outgrowth and synapse formation in cocultures of primary astrocytes and neurons under similar temporal conditions that μ opioids reduced TSP1 protein levels in astrocytes. Here we found that, after the same 8 day treatment, morphine or DAMGO diminished TSP2 protein levels in astrocytes. Therefore, μ opioids may deter synaptogenesis via both TSP1/2 isoforms, but by distinct mechanisms. KEYWORDS: Opioids, morphine, opioid receptors, astrocytes, ERK/MAPK, growth factors A strocytes make integral contributions to neuronal signaling in the brain by releasing molecules that act at synapses during different stages of synaptogenesis. These phases include pre-and postsynaptic development, function, maintenance, and plasticity.1−7 One family of key astrocyte-secreting molecules includes thrombospondins (TSPs), oligomeric, multidomain glycoproteins that promote synapse formation in vivo and in vitro.8−14 Yet another molecule is TGFβ that induces excitatory synapses at least in part via a D-serine dependent mechanism. 15 Interestingly, TGFβ and TSPs maintain a reciprocal relationship in astrocytes. Early on, it was recognized that TSP1 activates TGFβ. 16 Alternatively, several independent research groups have reported that in astrocytes TGFβ increases TSP1 gene expression and/or protein levels as shown by immunoblotting, qRT-PCR, and in situ hybridization.17−20 Both of these processes appear to be initiated in the extrac...

Role of GPCR (mu-opioid)–receptor tyrosine kinase (epidermal growth factor) crosstalk in opioid-induced hyperalgesic priming (type II)

“…In this study, we investigated the mechanisms mediating the induction of OIH and the prolongation of PGE 2 -induced hyperalgesia in Type II hyperalgesic priming. In particular, we examined the role of the interaction of MOR, a Gα i -protein coupled receptor (GPCR) with epidermal growth factor receptor (EGFR), a receptor tyrosine kinase (RTK) that has been demonstrated to have crosstalk with opioid receptors [10; 16; 42]. We also investigated second messenger, downstream of MOR, involved in the induction of OIH and prolongation of PGE 2 hyperalgesia in Type II hyperalgesic priming.…”

Macroglia-derived thrombospondin 2 regulates alterations of presynaptic proteins of retinal neurons following elevated hydrostatic pressure

“…Glia cells have been shown to be able to be activated in neuropathic conditions, and the activated glia cells may release reactive factors to promote synaptogenesis in the developed nervous system. Then, the reactive synaptogenesis may result in various nervous system diseases [ 15 , 20 , 21 ]. Lo[ 41 ] found that, after neonatal unilateral transection of the infraorbital branch of the trigeminal nerve, glia cells were activated, demonstrated by cell proliferation and upregulation of GFAP, and accompanied by reactive synaptogenesis in the nerve projection zone.…”

“…Therefore, clearly further studies are needed to explore the synaptogenic roles of different increased TSPs after injury. It was also worthy to note that the microglia cells are also the source of TSP2[ 20 ]. The relatively small percentage of microglia cells is approximately 4% in retinal cell cultures.…”

“…In recent years, glia cells have been thought to be active modulators in developing synaptogenesis [ 18 , 19 ]. In many neuropathic and nervous injury states, glia cells can be activated, characterized by increased proliferation and glial fibrillary acidic protein (GFAP) expression[ 20 , 21 ]. Our previous study also demonstrated that retinal synaptic changes are accompanied by activation of macroglia cells (astrocytes and Müller cells) following acute elevated IOP[ 22 ].…”

“…The TSP family consists of five isoforms (TSP1/2/3/4/5)[ 25 ], which are detectable in the developing and adult CNS [ 26 ]. Most notably, TSP1 and TSP2 are the most common types in the nervous system[ 20 , 27 ]. Our previous study found that the distributions of TSP1 and TSP2 in the rat retina are apparently distinct[ 28 ].…”