Molecular mechanisms of mutant mu-opioid receptors where naloxone acts as an agonist

Pain management is often one of the most difficult aspects of treatment for patients suffering from acute or chronic pain. The mu-opioid receptor (MOR) agonist, morphine, and its derivatives are highly used in pain management strategies. However, these medications have many side effects including respiratory depression, gastrointestinal problems, as well as dependence and addiction liabilities. For these reasons, innovative new modalities for pain management continue to be needed. One new approach to the design of opioid therapies for chronic pain with reduced liabilities is a targeted-gene therapy strategy developed by the lab of Dr. Ping-Yee Law at the University of Minnesota. This strategy makes novel use of a MOR S4.54A mutant at which the classical opioid antagonist, naloxone, acts as a partial agonist. Targeted gene therapy studies using this mutant have shown that naloxone becomes an antinociceptive agent at the S4.54A mutant both in vitro and in vivo. Because expression of the mutant MOR is targeted to the spinal cord injection site region, systemic administration of naloxone results in antagonism of all other (native) MOR's. The reduced number of receptors activated in this paradigm results in no measurable dependence/addiction as seen with traditional mu agonists like morphine. Despite the clear success of basing this strategy on the S4.54A MOR mutant, the origins of this unusual phenotype are not yet understood. It was therefore the overall goal of this dissertation to identify the molecular basis for the agonism of naloxone at this novel S4.54A mutant. To this end, a model of the wild-type and S4.54A mu opioid receptor was developed and ligand docking studies were used to probe this model. The opioid receptors, delta, kappa and mu, belong to the Class A subfamily of G-Protein Coupled Receptors (GPCRs). These are integral membrane proteins that possess seven transmembrane helices (TMHs) arranged to form a closed bundle with loops that extend both extracellularly and intracellularly. The N-terminus is extracellular and the C-terminus is intracellular. In recent years, X-ray crystallography studies have yielded structures of numerous GPCRs. In 2012, the nociception/orphanin FQ receptor and the mu, delta and kappa opioid receptor crystal structures were published. Prior to the release of the MOR crystal structure, we developed a homology model of WT MOR using the β2-AR crystal structure2,3 as a template with substitutions for TMH 1, 2, 4 and 7 based on sequence divergences, as described in methods. This model was then used for studies of the MOR including analyzing the receptor for cholesterol and palmitoylaiton interactions as well as modeling a homodimer interface for the MOR based on experimental data. In 2012, new models of WT MOR and the S4.54A/L mutants were developed using the MOR crystal structure. The conformational change in TMH4 that would be created upon the S4.54A mutation was examined using the simulated annealing/Monte Carlo method, Conformational Memories, and the result was incorporated into the model. The S4.54A mutant model was then used for naloxone docking studies using Glide. These studies revealed that in the crystal structure, Y3.34 forms a hydrogen bond with the sidechain of S4.54; however, in the S4.54A MT MOR, this interaction is broken as there is no polar partner for Y3.34. The breaking of this interaction allows the extracellular end of TMH4 to kink away from TMH3 and towards TMH5, which leads to changes in the packing of the receptor binding pocket. In the wild type MOR, naloxone interacts with D3.32 and sits in close proximity to the binding pocket "toggle switch" residue, W6.48, restricting its movement. However, in the S4.54A MT MOR, naloxone sits higher in the binding pocket, away from W6.48 and interacts with D3.32 and E5.35. In this higher location, naloxone exerts no effect on W6.48, permitting W6.48 to assume an active state conformation. This shift in binding pocket location for naloxone may be the origin of naloxone's partial agonism in the S4.54A MOR mutant. We also explored additional experimental data generated in Dr. Ping Law's lab for other mutations at the 4.54 locus. Mutating S4.54 to Phe or Gly results in the same phenotype as the S4.54A mutation. On the other hand, for Ile or Val mutants, naloxone behaves as in WT MOR. We propose that in the case of the S4.54 I / V, an increase in hydrophobic interactions between W4.50 and I/V4.54 allow TMH4 to maintain its wild type conformation. However, while the S4.54F is also able to increase hydrophobic interactions, its size prevents the helix from maintaining the wild type shape. In the S4.54L mutant, there is no increase in hydrophobic interactions and the orientation of the leucine gives rise to a straighter TMH4, as seen in the S4.54A MT MOR. The S4.54G mutant offers additional flexibility and a higher turn ratio, with 5 residues per turn in that region such that the extracellular end of TMH4 moves away from TMH3 and towards TMH5. Additionally, Law and coworkers have published studies using a S4.54L/T7.44A/C7.47S triple mutant MOR that gives rise to naloxone acting as a full agonist.4 While this gene therapy has been shown in cells and in spinal cord, the underlying mechanism is unknown. A triple mutant MOR model was developed and analyzed to determine the molecular mechanism for which naloxone acts as an agonist. The binding pocket for mu opioid ligands is formed by TMHs 3, 5 and 6 in the wild type receptor, as seen in the crystal structure with β-FNA5 and in our glide dock of naloxone (see Chapter 3). As studied in the single mutant MOR, S4.54 is a lipid facing residue. Interestingly, both of the mutated residues on TMH7 (T7.44 and C7.47) in the triple mutant MOR also face lipid. We report here that the combination of the S4.54L mutation on TMH4 along with TMH7 face shift changes occur upon mutation of T7.44 and C7.47 produce overall packing changes that give rise to a different binding pocket than seen in the wild type or single mutant MORs. These changes result in naloxone's ability to fully activate the S4.54L/T7.44A/C7.47S MOR

Palmitoylation and membrane cholesterol stabilize μ-opioid receptor homodimerization and G protein coupling

<p>Abstract</p> <p>Background</p> <p>A cholesterol-palmitoyl interaction has been reported to occur in the dimeric interface of the β₂-adrenergic receptor crystal structure. We sought to investigate whether a similar phenomenon could be observed with μ-opioid receptor (OPRM1), and if so, to assess the role of cholesterol in this class of G protein-coupled receptor (GPCR) signaling.</p> <p>Results</p> <p>C3.55(170) was determined to be the palmitoylation site of OPRM1. Mutation of this Cys to Ala did not affect the binding of agonists, but attenuated receptor signaling and decreased cholesterol associated with the receptor signaling complex. In addition, both attenuation of receptor palmitoylation (by mutation of C3.55[170] to Ala) and inhibition of cholesterol synthesis (by treating the cells with simvastatin, a HMG-CoA reductase inhibitor) impaired receptor signaling, possibly by decreasing receptor homodimerization and Gαi2 coupling; this was demonstrated by co-immunoprecipitation, immunofluorescence colocalization and fluorescence resonance energy transfer (FRET) analyses. A computational model of the OPRM1 homodimer structure indicated that a specific cholesterol-palmitoyl interaction can facilitate OPRM1 homodimerization at the TMH4-TMH4 interface.</p> <p>Conclusions</p> <p>We demonstrate that C3.55(170) is the palmitoylation site of OPRM1 and identify a cholesterol-palmitoyl interaction in the OPRM1 complex. Our findings suggest that this interaction contributes to OPRM1 signaling by facilitating receptor homodimerization and G protein coupling. This conclusion is supported by computational modeling of the OPRM1 homodimer.</p

PPARα is essential for retinal lipid metabolism and neuronal survival

Background: Peroxisome proliferator activated receptor-alpha (PPARα) is a ubiquitously expressed nuclear receptor. The role of endogenous PPARα in retinal neuronal homeostasis is unknown. Retinal photoreceptors are the highest energy-consuming cells in the body, requiring abundant energy substrates. PPARα is a known regulator of lipid metabolism, and we hypothesized that it may regulate lipid use for oxidative phosphorylation in energetically demanding retinal neurons. Results: We found that endogenous PPARα is essential for the maintenance and survival of retinal neurons, with Pparα -/- mice developing retinal degeneration first detected at 8 weeks of age. Using extracellular flux analysis, we identified that PPARα mediates retinal utilization of lipids as an energy substrate, and that ablation of PPARα ultimately results in retinal bioenergetic deficiency and neurodegeneration. This may be due to PPARα regulation of lipid transporters, which facilitate the internalization of fatty acids into cell membranes and mitochondria for oxidation and ATP production. Conclusion: We identify an endogenous role for PPARα in retinal neuronal survival and lipid metabolism, and furthermore underscore the importance of fatty acid oxidation in photoreceptor survival. We also suggest PPARα as a putative therapeutic target for age-related macular degeneration, which may be due in part to decreased mitochondrial efficiency and subsequent energetic deficits. Electronic supplementary material The online version of this article (doi:10.1186/s12915-017-0451-x) contains supplementary material, which is available to authorized users

The attitudes, beliefs and behaviours of GPs regarding exercise for chronic knee pain: a systematic review

<p>Abstract</p> <p>Background</p> <p>Joint pain, specifically chronic knee pain (CKP), is a frequent cause of chronic pain and limitation of function and mobility among older adults. Multiple evidence-based guidelines recommend exercise as a first-line treatment for all patients with CKP or knee osteoarthritis (KOA), yet healthcare practitioners' attitudes and beliefs may limit their implementation. This systematic review aims to identify the attitudes, beliefs and behaviours of General Practitioners (GPs) regarding the use of exercise for CKP/KOA.</p> <p>Methods</p> <p>We searched four electronic databases between inception and January 2008, using subject headings to identify studies examining the attitudes, beliefs or behaviours of GPs regarding the use of exercise for the treatment of CKP/KOA in adults aged over 45 years in primary care. Studies referring to patellofemoral pain syndrome or CKP secondary to other causes or that occurring in a prosthetic joint were excluded. Once inclusion and exclusion criteria were applied, study data were extracted and summarised. Study quality was independently reviewed using two assessment tools.</p> <p>Results</p> <p>From 2135 potentially relevant articles, 20 were suitable for inclusion. A variety of study methodologies and approaches to measuring attitudes beliefs and behaviours were used among the studies. Quality assessment revealed good reporting of study objective, type, outcome factors and, generally, the sampling frame. However, criticisms included use of small sample sizes, low response rates and under-reporting of non-responder factors. Although 99% of GPs agreed that exercise should be used for CKP/KOA and reported ever providing advice or referring to a physiotherapist, up to 29% believed that rest was the optimum management approach. The frequency of actual provision of exercise advice or physiotherapy referral was lower. Estimates of provision of exercise advice and physiotherapy referral were generally higher for vignette-based studies (exercise advice 9%-89%; physiotherapy referral 44%-77%) than reviews of actual practice (exercise advice 5%-52%; physiotherapy referral 13-63%). <it>A</it><it>dvice to exercise </it>and exercise <it>prescription </it>were not clearly differentiated.</p> <p>Conclusions</p> <p>Attitudes and beliefs of GPs towards exercise for CKP/KOA vary widely and exercise appears to be underused in the management of CKP/KOA. Limitations of the evidence base include the paucity of studies directly examining attitudes of GPs, poor methodological quality, limited generalisability of results and ambiguity concerning GPs' expected roles. Further investigation is required of the roles of GPs in using exercise as first-line management of CKP/KOA.</p

The Protective Role of Microglial PPARα in Diabetic Retinal Neurodegeneration and Neurovascular Dysfunction

Microglial activation and subsequent pathological neuroinflammation contribute to diabetic retinopathy (DR). However, the underlying mechanisms of microgliosis, and means to effectively suppress pathological microgliosis, remain incompletely understood. Peroxisome proliferator-activated receptor alpha (PPARα) is a transcription factor that regulates lipid metabolism. The present study aimed to determine if PPARα affects pathological microgliosis in DR. In global Pparα mice, retinal microglia exhibited decreased structural complexity and enlarged cell bodies, suggesting microglial activation. Microglia-specific conditional Pparα−/− (PCKO) mice showed decreased retinal thickness as revealed by optical coherence tomography. Under streptozotocin (STZ)-induced diabetes, diabetic PCKO mice exhibited decreased electroretinography response, while diabetes-induced retinal dysfunction was alleviated in diabetic microglia-specific Pparα-transgenic (PCTG) mice. Additionally, diabetes-induced retinal pericyte loss was exacerbated in diabetic PCKO mice and alleviated in diabetic PCTG mice. In cultured microglial cells with the diabetic stressor 4-HNE, metabolic flux analysis demonstrated that Pparα ablation caused a metabolic shift from oxidative phosphorylation to glycolysis. Pparα deficiency also increased microglial STING and TNF-α expression. Taken together, these findings revealed a critical role for PPARα in pathological microgliosis, neurodegeneration, and vascular damage in DR, providing insight into the underlying molecular mechanisms of microgliosis in this context and suggesting microglial PPARα as a potential therapeutic target

CaMKII kinase activity, targeting and control of cellular functions: effect of single and double phosphorylation of CaMKIIα

Calcium/calmodulin-stimulated protein kinase II (CaMKII) is a ubiquitously expressed multifunctional kinase, which regulates many cellular processes, including synaptic plasticity and proliferation. CaMKII is activated by binding calmodulin (CaM), triggered by an increase in intracellular Ca²⁺. CaMKII can autophosphorylate at several residues, including T253, T286, and T305/306, which alters CaMKII activity and targeting in different ways. Phosphorylation at T286 induces autonomous activity, whereas phosphorylation at T305/306 prevents CaMKII activation. Phosphorylation at T253 has no effect on CaMKII activity in vitro. Phosphorylation at each of these sites changes the subcellular location of CaMKII, and alters protein interactions. To investigate the mechanisms by which dual phosphorylation of CaMKII might regulate cellular functions we examined the effects of double phosphomimic mutation (at T253D/T286D or T286D/T305D) on kinase activity and targeting. We showed that both double phosphomimic mutations altered targeting whereas only T286D/T305D altered kinase activity in vitro. We also showed that overexpressing either T253D/T286D or T286D/T305D altered cell proliferation rates, and that this effect was different from the effects observed with the relevant single phosphomimic mutation. These results indicate the importance of targeting as a regulatory mechanism in CaMKII control of cell function

Neuroprotective effects of PPARα in retinopathy of type 1 diabetes.

Diabetic retinopathy (DR) is a common neurovascular complication of type 1 diabetes. Current therapeutics target neovascularization characteristic of end-stage disease, but are associated with significant adverse effects. Targeting early events of DR such as neurodegeneration may lead to safer and more effective approaches to treatment. Two independent prospective clinical trials unexpectedly identified that the PPARα agonist fenofibrate had unprecedented therapeutic effects in DR, but gave little insight into the physiological and molecular mechanisms of action. The objective of the present study was to evaluate potential neuroprotective effects of PPARα in DR, and subsequently to identify the responsible mechanism of action. Here we reveal that activation of PPARα had a robust protective effect on retinal function as shown by Optokinetic tracking in a rat model of type 1 diabetes, and also decreased retinal cell death, as demonstrated by a DNA fragmentation ELISA. Further, PPARα ablation exacerbated diabetes-induced decline of visual function as demonstrated by ERG analysis. We further found that PPARα improved mitochondrial efficiency in DR, and decreased ROS production and cell death in cultured retinal neurons. Oxidative stress biomarkers were elevated in diabetic Pparα-/- mice, suggesting increased oxidative stress. Mitochondrially mediated apoptosis and oxidative stress secondary to mitochondrial dysfunction contribute to neurodegeneration in DR. Taken together, these findings identify a robust neuroprotective effect for PPARα in DR, which may be due to improved mitochondrial function and subsequent alleviation of energetic deficits, oxidative stress and mitochondrially mediated apoptosis