The effects of chronic high-dose morphine on microgliosis and the microglial transcriptome in rat spinal cord.

Background: Opioids are efficacious and safe analgesic drugs in short-term use for acute pain but chronic use can lead to tolerance and dependence. Opioid-induced microglial activation may contribute to the development of tolerance and this process may differ between males and females. A link is suggested between this microglial activation and inflammation, disturbances of circadian rhythms, and neurotoxic effects. We set out to further delineate the effects of chronic morphine on pain behaviour, microglial and neuronal staining, and the transcriptome of spinal microglia, to better understand the role of microglia in the consequences of long-term high-dose opioid administration. Experimental Approach: In two experiments, we administered increasing subcutaneous doses of morphine hydrochloride or saline to male and female rats. Thermal nociception was assessed with the tail flick and hot plate tests. In Experiment I, spinal cord (SC) samples were prepared for immunohistochemical staining for microglial and neuronal markers. In Experiment II, the transcriptome of microglia from the lumbar SC was analysed. Key Results: Female and male rats had similar antinociceptive responses to morphine and developed similar antinociceptive tolerance to thermal stimuli following chronic increasing high doses of s.c. morphine. The area of microglial IBA1-staining in SC decreased after 2 weeks of morphine administration in both sexes. Following morphine treatment, the differentially expressed genes identified in the microglial transcriptome included ones related to the circadian rhythm, apoptosis, and immune system processes. Conclusions: Female and male rats showed similar pain behaviour following chronic high doses of morphine. This was associated with decreased staining of spinal microglia, suggesting either decreased activation or apoptosis. High-dose morphine administration also associated with several changes in gene expression in SC microglia, e.g., those related to the circadian rhythm (Per2, Per3, Dbp). These changes should be considered in the clinical consequences of long-term high-dose administration of opioids.

Seipin regulates ER-lipid droplet contacts and cargo delivery.

Seipin is an endoplasmic reticulum (ER) membrane protein implicated in lipid droplet (LD) biogenesis and mutated in severe congenital lipodystrophy (BSCL2). Here, we show that seipin is stably associated with nascent ER-LD contacts in human cells, typically via one mobile focal point per LD Seipin appears critical for such contacts since ER-LD contacts were completely missing or morphologically aberrant in seipin knockout and BSCL2 patient cells. In parallel, LD mobility was increased and protein delivery from the ER to LDs to promote LD growth was decreased. Moreover, while growing LDs normally acquire lipid and protein constituents from the ER, this process was compromised in seipin-deficient cells. In the absence of seipin, the initial synthesis of neutral lipids from exogenous fatty acid was normal, but fatty acid incorporation into neutral lipids in cells with pre-existing LDs was impaired. Together, our data suggest that seipin helps to connect newly formed LDs to the ER and that by stabilizing ER-LD contacts seipin facilitates the incorporation of protein and lipid cargo into growing LDs in human cells.

OSBP-related protein-2 (ORP2): a novel Akt effector that controls cellular energy metabolism.

ORP2 is a ubiquitously expressed OSBP-related protein previously implicated in endoplasmic reticulum (ER)-lipid droplet (LD) contacts, triacylglycerol (TG) metabolism, cholesterol transport, adrenocortical steroidogenesis, and actin-dependent cell dynamics. Here, we characterize the role of ORP2 in carbohydrate and lipid metabolism by employing ORP2-knockout (KO) hepatoma cells (HuH7) generated by CRISPR-Cas9 gene editing. The ORP2-KO and control HuH7 cells were subjected to RNA sequencing, analyses of Akt signaling, carbohydrate and TG metabolism, the extracellular acidification rate, and the lipidome, as well as to transmission electron microscopy. The loss of ORP2 resulted in a marked reduction of active phosphorylated Akt(Ser473) and its target Glycogen synthase kinase 3ß(Ser9), consistent with defective Akt signaling. ORP2 was found to form a physical complex with the key controllers of Akt activity, Cdc37, and Hsp90, and to co-localize with Cdc37 and active Akt(Ser473) at lamellipodial plasma membrane regions, in addition to the previously reported ER-LD localization. ORP2-KO reduced glucose uptake, glycogen synthesis, glycolysis, mRNA-encoding glycolytic enzymes, and SREBP-1 target gene expression, and led to defective TG synthesis and storage. ORP2-KO did not reduce but rather increased ER-LD contacts under basal culture conditions and interfered with their expansion upon fatty acid loading. Together with our recently published work (Kentala et al. in FASEB J 32:1281-1295, 2018), this study identifies ORP2 as a new regulatory nexus of Akt signaling, cellular energy metabolism, actin cytoskeletal function, cell migration, and proliferation.

Identification of selective cytotoxic and synthetic lethal drug responses in triple negative breast cancer cells.

BACKGROUND: Triple negative breast cancer (TNBC) is a highly heterogeneous and aggressive type of cancer that lacks effective targeted therapy. Despite detailed molecular profiling, no targeted therapy has been established. Hence, with the aim of gaining deeper understanding of the functional differences of TNBC subtypes and how that may relate to potential novel therapeutic strategies, we studied comprehensive anticancer-agent responses among a panel of TNBC cell lines. METHOD: The responses of 301 approved and investigational oncology compounds were measured in 16 TNBC cell lines applying a functional profiling approach. To go beyond the standard drug viability effect profiling, which has been used in most chemosensitivity studies, we utilized a multiplexed readout for both cell viability and cytotoxicity, allowing us to differentiate between cytostatic and cytotoxic responses. RESULTS: Our approach revealed that most single-agent anti-cancer compounds that showed activity for the viability readout had no or little cytotoxic effects. Major compound classes that exhibited this type of response included anti-mitotics, mTOR, CDK, and metabolic inhibitors, as well as many agents selectively inhibiting oncogene-activated pathways. However, within the broad viability-acting classes of compounds, there were often subsets of cell lines that responded by cell death, suggesting that these cells are particularly vulnerable to the tested substance. In those cases we could identify differential levels of protein markers associated with cytotoxic responses. For example, PAI-1, MAPK phosphatase and Notch-3 levels associated with cytotoxic responses to mitotic and proteasome inhibitors, suggesting that these might serve as markers of response also in clinical settings. Furthermore, the cytotoxicity readout highlighted selective synergistic and synthetic lethal drug combinations that were missed by the cell viability readouts. For instance, the MEK inhibitor trametinib synergized with PARP inhibitors. Similarly, combination of two non-cytotoxic compounds, the rapamycin analog everolimus and an ATP-competitive mTOR inhibitor dactolisib, showed synthetic lethality in several mTOR-addicted cell lines. CONCLUSIONS: Taken together, by studying the combination of cytotoxic and cytostatic drug responses, we identified a deeper spectrum of cellular responses both to single agents and combinations that may be highly relevant for identifying precision medicine approaches in TNBC as well as in other types of cancers.

Target inhibition networks: predicting selective combinations of druggable targets to block cancer survival pathways.

A recent trend in drug development is to identify drug combinations or multi-target agents that effectively modify multiple nodes of disease-associated networks. Such polypharmacological effects may reduce the risk of emerging drug resistance by means of attacking the disease networks through synergistic and synthetic lethal interactions. However, due to the exponentially increasing number of potential drug and target combinations, systematic approaches are needed for prioritizing the most potent multi-target alternatives on a global network level. We took a functional systems pharmacology approach toward the identification of selective target combinations for specific cancer cells by combining large-scale screening data on drug treatment efficacies and drug-target binding affinities. Our model-based prediction approach, named TIMMA, takes advantage of the polypharmacological effects of drugs and infers combinatorial drug efficacies through system-level target inhibition networks. Case studies in MCF-7 and MDA-MB-231 breast cancer and BxPC-3 pancreatic cancer cells demonstrated how the target inhibition modeling allows systematic exploration of functional interactions between drugs and their targets to maximally inhibit multiple survival pathways in a given cancer type. The TIMMA prediction results were experimentally validated by means of systematic siRNA-mediated silencing of the selected targets and their pairwise combinations, showing increased ability to identify not only such druggable kinase targets that are essential for cancer survival either individually or in combination, but also synergistic interactions indicative of non-additive drug efficacies. These system-level analyses were enabled by a novel model construction method utilizing maximization and minimization rules, as well as a model selection algorithm based on sequential forward floating search. Compared with an existing computational solution, TIMMA showed both enhanced prediction accuracies in cross validation as well as significant reduction in computation times. Such cost-effective computational-experimental design strategies have the potential to greatly speed-up the drug testing efforts by prioritizing those interventions and interactions warranting further study in individual cancer cases.

Gene expression in the dorsal root ganglion and the cerebrospinal fluid metabolome in polyneuropathy and opioid tolerance in rats.

First-line pharmacotherapy for peripheral neuropathic pain (NP) of diverse pathophysiology consists of antidepressants and gabapentinoids, but only a minority achieve sufficient analgesia with these drugs. Opioids are considered third-line analgesics in NP due to potential severe and unpredictable adverse effects in long-term use. Also, opioid tolerance and NP may have shared mechanisms, raising further concerns about opioid use in NP. We set out to further elucidate possible shared and separate mechanisms after chronic morphine treatment and oxaliplatin-induced and diabetic polyneuropathies, and to identify potential diagnostic markers and therapeutic targets. We analysed thermal nociceptive behaviour, the transcriptome of dorsal root ganglia (DRG) and the metabolome of cerebrospinal fluid (CSF) in these three conditions, in rats. Several genes were differentially expressed, most following oxaliplatin and least after chronic morphine treatment, compared with saline-treated rats. A few genes were differentially expressed in the DRGs in all three models (e.g. Csf3r and Fkbp5). Some, e.g. Alox15 and Slc12a5, were differentially expressed in both diabetic and oxaliplatin models. Other differentially expressed genes were associated with nociception, inflammation, and glial cells. The CSF metabolome was most significantly affected in the diabetic rats. Interestingly, we saw changes in nicotinamide metabolism, which has been associated with opioid addiction and withdrawal, in the CSF of morphine-tolerant rats. Our results offer new hypotheses for the pathophysiology and treatment of NP and opioid tolerance. In particular, the role of nicotinamide metabolism in opioid addiction deserves further study.

dGRASP-mediated noncanonical integrin secretion is required for Drosophila epithelial remodeling.

Integral plasma membrane proteins are typically transported in the secretory pathway from the endoplasmic reticulum and the Golgi complex. Here we show that at specific stages of Drosophila development corresponding to morphological changes in epithelia, apposed basolateral membranes separate slightly, allowing new plasma membrane contacts with basal extracellular matrix. At these sites, newly synthesized integrin alpha subunits are deposited via a mechanism that appears to bypass the Golgi. We show that the Drosophila Golgi resident protein dGRASP localizes to these membrane domains and that, in the absence of dGRASP, the integrin subunit is retained intracellularly in both follicular and wing epithelia that are found disrupted. We propose that this dGRASP-mediated noncanonical secretion route allows for developmental regulation of integrin function upon epithelial remodeling. We speculate that this mechanism might be used during development as a means of targeting a specific subset of transmembrane proteins to the plasma membrane.

Seipin localizes at endoplasmic-reticulum-mitochondria contact sites to control mitochondrial calcium import and metabolism in adipocytes.

Deficiency of the endoplasmic reticulum (ER) protein seipin results in generalized lipodystrophy by incompletely understood mechanisms. Here, we report mitochondrial abnormalities in seipin-deficient patient cells. A subset of seipin is enriched at ER-mitochondria contact sites (MAMs) in human and mouse cells and localizes in the vicinity of calcium regulators SERCA2, IP3R, and VDAC. Seipin association with MAM calcium regulators is stimulated by fasting-like stimuli, while seipin association with lipid droplets is promoted by lipid loading. Acute seipin removal does not alter ER calcium stores but leads to defective mitochondrial calcium import accompanied by a widespread reduction in Krebs cycle metabolites and ATP levels. In mice, inducible seipin deletion leads to mitochondrial dysfunctions preceding the development of metabolic complications. Together, these data suggest that seipin controls mitochondrial energy metabolism by regulating mitochondrial calcium influx at MAMs. In seipin-deficient adipose tissue, reduced ATP production compromises adipocyte properties, contributing to lipodystrophy pathogenesis.

Role for formin-like 1-dependent acto-myosin assembly in lipid droplet dynamics and lipid storage.

Lipid droplets (LDs) are cellular organelles specialized in triacylglycerol (TG) storage undergoing homotypic clustering and fusion. In non-adipocytic cells with numerous LDs this is balanced by poorly understood droplet dissociation mechanisms. We identify non-muscle myosin IIa (NMIIa/MYH-9) and formin-like 1 (FMNL1) in the LD proteome. NMIIa and actin filaments concentrate around LDs, and form transient foci between dissociating LDs. NMIIa depletion results in decreased LD dissociations, enlarged LDs, decreased hydrolysis and increased storage of TGs. FMNL1 is required for actin assembly on LDs in vitro and for NMIIa recruitment to LDs in cells. We propose a novel acto-myosin structure regulating lipid storage: FMNL1-dependent assembly of myosin II-functionalized actin filaments on LDs facilitates their dissociation, thereby affecting LD surface-to-volume ratio and enzyme accessibility to TGs. In neutrophilic leucocytes from MYH9-related disease patients NMIIa inclusions are accompanied by increased lipid storage in droplets, suggesting that NMIIa dysfunction may contribute to lipid imbalance in man.

Systematic Mapping of Kinase Addiction Combinations in Breast Cancer Cells by Integrating Drug Sensitivity and Selectivity Profiles.

Chemical perturbation screens offer the possibility to identify actionable sets of cancer-specific vulnerabilities. However, most inhibitors of kinases or other cancer targets result in polypharmacological effects, which complicate the identification of target dependencies directly from the drug-response phenotypes. In this study, we developed a chemical systems biology approach that integrates comprehensive drug sensitivity and selectivity profiling to provide functional insights into both single and multi-target oncogenic signal addictions. When applied to 21 breast cancer cell lines, perturbed with 40 kinase inhibitors, the subtype-specific addiction patterns clustered in agreement with patient-derived subtypes, while showing considerable variability between the heterogeneous breast cancers. Experimental validation of the top predictions revealed a number of co-dependencies between kinase targets that led to unexpected synergistic combinations between their inhibitors, such as dasatinib and axitinib in the triple-negative basal-like HCC1937 cell line.

Integrins mediate their unconventional, mechanical-stress-induced secretion via RhoA and PINCH in Drosophila.

During the epithelium remodelling such as the flattening of the Drosophila follicular epithelium, the alpha-integrin subunits are unconventionally secreted through a dGRASP-dependent route that is built de novo. The biogenetic process starts with the upregulation of a small subset of targeted mRNAs, including dgrasp. Here, we show that dgrasp mRNA upregulation is triggered by the tension of the underlying oocyte and by applied external forces at the basal side of the follicular epithelium. We show that integrins are also involved in dgrasp mRNA upregulation and the epithelium remodelling. Tension leads to the recruitment of RhoA to the plasma membrane, where it participates in its remodelling. The LIM protein PINCH can cycle to the nucleus and is involved in dgrasp mRNA upregulation. We propose that integrins are involved in triggering the biogenesis of their own unconventional secretion route that they use to strengthen adhesion and ensure epithelial integrity at the next stages of development, perhaps by acting as mechanosensors of the underlying tension through RhoA and PINCH.

Endoplasmic reticulum exit of a secretory glycoprotein in the absence of sec24p family proteins in yeast.

Glycoproteins exit the endoplasmic reticulum (ER) of the yeast Saccharomyces cerevisiae in coat protein complex II (COPII) coated vesicles. The coat consists of the essential proteins Sec23p, Sec24p, Sec13p, Sec31p, Sar1p and Sec16p. Sec24p and its two nonessential homologues Sfb2p and Sfb3p have been suggested to serve in cargo selection. Using temperature-sensitive sec24-1 mutants, we showed previously that a secretory glycoprotein, Hsp150, does not require functional Sec24p for ER exit. Deletion of SFB2, SFB3 or both from wild type or the deletion of SFB2 from sec24-1 cells did not affect Hsp150 transport. SFB3 deletion has been reported to be lethal in sec24-1. However, here we constructed a sec24-1 Deltasfb3 and a sec24-1 Deltasfb2 Deltasfb3 strain and show that Hsp150 was secreted slowly in both. Turning off the SEC24 gene did not inhibit Hsp150 secretion either, and the lack of SEC24 expression in a Deltasfb2 Deltasfb3 deletant still allowed some secretion. The sec24-1 Deltasfb2 Deltasfb3 mutant grew slower than sec24-1. The cells were irregularly shaped, budded from random sites and contained proliferated ER at permissive temperature. At restrictive temperature, the ER formed carmellae-like proliferations. Our data indicate that ER exit may occur in vesicles lacking a full complement of Sec23p/24p and Sec13p/31p, demonstrating diversity in the composition of the COPII coat.

Activity of recycling Golgi mannosyltransferases in the yeast endoplasmic reticulum.

In yeast primary N- and O-glycans are attached to proteins in the endoplasmic reticulum (ER), and they are elongated in the Golgi. Thus, glycan extension by Golgi enzymes has been taken as evidence for arrival of a protein in the Golgi. Two alpha 1,6-mannosyltransferase activity-containing multiprotein complexes have been reported to recycle between the Golgi and the ER, but since resident ER proteins are not Golgi-modified, Golgi enzymes were not thought to function in the ER. Here we show that when protein exit from the ER was blocked in COPII-defective yeast mutants, the N-glycans of vacuolar carboxypeptidase Y and a set of unidentified glycoproteins were decorated with an alpha 1,6-mannose residue, normally added in the Golgi by Och1p. Immunofluorescent staining demonstrated that Och1p accumulated in the ER under these conditions. Concomitantly, primary O-glycans of a secretory protein were extended, apparently by the medial Golgi transferase Mnt1p. Similar O-glycan extension occurred in wild-type cells when an HDEL-tagged protein was allowed to encounter glycosyltransferases in the Golgi during recycling between ER and Golgi. Golgi-specific glycosylation in the ER was reduced when Golgi-to-ER traffic was blocked, confirming that glycan extension in the ER was mainly due to recycling, rather than newly synthesized transferases.

Active and specific recruitment of a soluble cargo protein for endoplasmic reticulum exit in the absence of functional COPII component Sec24p.

Exit of proteins from the yeast endoplasmic reticulum (ER) is thought to occur in vesicles coated by four proteins, Sec13p, Sec31p, Sec23p and Sec24p, which assemble at ER exit sites to form the COPII coat. Sec13p may serve a structural function, whereas Sec24p has been suggested to operate in selection of cargo proteins into COPII vesicles. We showed recently that the soluble glycoprotein Hsp150 exited the ER in the absence of Sec13p function. Here we show that its ER exit did not require functional Sec24p. Hsp150 was secreted to the medium in a sec24-1 mutant at restrictive temperature 37 degrees C, while cell wall invertase and vacuolar carboxypeptidase Y remained in the ER. The determinant guiding Hsp150 to this transport route was mapped to the C-terminal domain of 114 amino acids by deletion analysis, and by an HRP fusion protein-based EM technology adapted here for yeast. This domain actively mediated ER exit of Sec24p-dependent invertase in the absence of Sec24p function. However, the domain was entirely dispensable for ER exit when Sec24p was functional. The Sec24p homolog Sfb2p was shown not to compensate for nonfunctional Sec24p in ER exit of Hsp150. Our data show that a soluble cargo protein, Hsp150, is selected actively and specifically to budding sites lacking normal Sec24p by a signature residing in its C-terminal domain.