Interactions between the opioid and serotonin systems in chronic pain : quantitative live cell study by fluorescence cross-correlation spectroscopy (FCCS)

Abstract

Chronic pain is a major health issue worldwide. It enacts considerable suffering on the affected individuals and significantly increases the societal burden, as it is the most common reason why individuals seek sick leave and ask for medical help. Pharmacotherapies for chronic pain are not satisfactory, and many patients, despite taking medications, do not find relief and end up using opioids. Long-term opioid use carries with it numerus adverse effects, including but not limited to opioid induced hyperalgesia (OIH), tolerance, dependence (addiction) and opioid-related deaths. OIH is defined as a state of nociceptive sensitization, i.e. a state of becoming more sensitive to painful stimuli when using opioids for a long time. Cellular and molecular mechanisms that underlie OIH are still not fully understood, and efficient treatment strategies that retain the analgesic effects of opioids, while reducing this negative side effect are therefore underdeveloped.

Clinical studies in healthy subjects and patients with fibromyalgia, a chronic pain syndrome with abnormalities in cerebral opioid signaling, have implicated interactions between the mu-opioid (MOP) and the serotonin 1A (5-HT1A) receptors in descending pain modulatory circuits as important for chronic pain modulation. Preclinical studies have shown that co-treatment with 5-HT1A agonists reduces OIH, diminishes the rewarding effects of morphine and the development of opioid tolerance. They have also provided indirect evidence for the co-localization of MOP and 5-HT1A receptors in the same nerve terminals, and demonstrated that MOP and 5-HT1A synergistically inhibited GABA release in the periaqueductal gray (PAG), a structure that mediates opioid-based pain control. Based on these findings, it was proposed that MOP and 5-HT1A heterodimerization is a potential mechanism through which MOP- and 5-HT1A-mediated signaling pathways are interlinked. It is further hypothesized that MOP and 5-HT1A heterodimer formation alters cellular signaling and contributes to neuroplastic changes that, eventually, lead to sensitization of pronociceptive pathways at the organism level. However, while co-localization of MOP and 5-HT1A receptors in discrete brain and spinal cord regions is well documented, the existence of heterodimers between MOP and 5-HT1A receptors has, thus far, only been shown in one study, which relied on the use of co-immunoprecipitation and Bioluminescence Resonance Energy Transfer (BRET) to demonstrate that these heterodimer complexes could form.

The primary objective of my PhD studies was to challenge the hypothesis that prolonged exposure to non-peptide opioids promotes heterodimer formation between MOP and 5-HT1A receptors, and that this effect can be abolished by co-treatment with 5-HT1A agonists. To this aim, cell lines of human and rat origin where genetically transformed to stably express physiologically relevant levels of MOP and 5-HT1A receptors tagged with spectrally distinct fluorescence reporters. Fluorescence Cross-Correlation Spectroscopy (FCCS), the dual color variant of the quantitative and nondestructive analytical technique called Fluorescence Correlation Spectroscopy (FCS), was used to characterize the receptor-receptor interactions in live cells. Additionally, confocal laser scanning microscopy (CLSM) was used to map the consequences of non-peptide opioid treatment on Ca2+ signaling dynamics and western blotting was applied to investigate signaling cross-talk via mitogen-activated protein kinases (MAPKs) p38 and the extracellular signal-regulated kinase (ERK1/2).

The work presented in this thesis, summarized in papers I-V, has contributed to better understanding of important basic cellular and molecular mechanisms that underlie signal transduction and material uptake across the plasma membrane. In particular, the work presented in papers I-III focuses on cellular and molecular mechanisms that underlie the development of OIH. In Paper I, we have shown that prolonged exposure to non-peptide opioids facilitates heterodimer formation between MOP and 5-HT1A receptors in live cells expressing physiologically relevant receptor levels. The extent of receptor heterodimerization was found to be both, opioid-specific and dose-dependent. Furthermore, we have shown that different opioids differently affected second messenger pathways, as indicated by differences in Ca2+ signaling dynamics and differential activation of p38 and ERK1/2 MAPKs. In Paper II, we have shown that 5-HT1A agonists such as buspirone and three newly identified buspirone analogs: B2, B3 and B5, can effectively reverse MOP–5-HT1A heterodimerization, thus counteracting the aversive effects of morphine. Importantly, this study, which brought together molecular modeling, virtual screening and advanced experimental tests in live cells, may in the future lead to the development of new drugs that target MOP and 5-HT1A heterodimer formation. In paper III, we have quantitatively characterized using FCS/FCCS and PhotoActivated Localization Microscopy (PALM) the nanoscale lateral dynamics and spatial organization of wild type MOP and its naturally occurring isoform (MOPN40D). We have shown that this non-synonymous single-nucleotide polymorphism (SNP) in the OPRM1 gene encoding the MOP, which is known to confer pain- and substance abuse-specific phenotypes at the organism level, significantly affected the lateral dynamics and organization of MOPN40D at the nanoscale level. In particular, we found that MOP-containing domains were larger and more densely populated than the MOPN40D harboring domains, with a small fraction of molecules residing outside of nanodomains. The opposite was found for MOPN40D. Moreover, we found that cholesterol depletion dynamically regulated the partitioning of MOP but not of MOPN40D, and observed that MOP and MOPN40D differ with respect to opioid peptide-induced internalization, with MOP being readily internalized together with the opioid peptide upon stimulation with β-endorphin, whereas MOPN40D showed lower internalization propensity and was typically not internalized together with the opioid peptide. These apparently subtle differences at the nanoscale level may, at least in part, explain why differences with respect to opioid dependence and analgesia are observed at the organism level.

Papers IV and V describe collaborative work where CLSM and/or FCS/FCCS were used to characterize translocation across the plasma membrane and cellular uptake. In Paper IV, we focus on substance delivery using cell-penetrating peptides as a vehicle. Using FCS/FCCS, we revealed the heterogeneity underlying self-assembly of cell-penetrating peptides and oligonucleotides, in this case small interfering RNA (siRNA), into large molecular complexes. We showed that peptide monomers, peptide self-aggregates and polydisperse peptide/cargo complexes coexist in solution and in live cell plasma membrane, which could explain why diverse cellular uptake mechanisms were simultaneously observed for cell-penetrating peptides-based delivery of cargo molecules. In Paper V, CLSM imaging was used to examine the role of α-Gal carbohydrate on protein uptake and degradation by immature monocyte-derived dendritic cells (iMDDCs), as a potential cellular and molecular mechanisms underlying the development of allergy to red meat. CLSM imaging revealed that presence of allergenic α-Gal epitopes on the protein surface significantly increases the uptake of the investigated model antigens, BSA-α-Gal and HSA-α-Gal, by in vitro cultured human iMDDCs, and showed that the taken up proteins are processed in endosomes.