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1.  Correlation between ventral striatal catecholamine content and nociceptive thresholds in neuropathic mice 
Neuropathic pain is characterized by persistent, intractable pain following damage or dysfunction of the nervous system. Analgesics that include central, rather than purely peripheral, targets are more effective when treating neuropathic pain, highlighting the spinal and/or supraspinal mechanisms that contribute to this aberrant pain condition. The striatum represents one of the brain regions that have been implicated in pain processing. Release of dopamine in the ventral striatum is normally associated with analgesia. Clinical and human imaging studies suggest dopamine is disrupted in neuropathic pain patients, although the conclusions drawn from these studies are limited by their non-invasive imaging or pharmacological approaches. In this study, we used a C57Bl/6 mouse model of neuropathic pain to describe the changes in neurotransmitter content in the striatum and their relationship to evoked pain thresholds. Striatal dopamine content negatively correlated with mechanical thresholds in sham animals. Neuropathic pain animals had reduced dopamine content that was not correlated with mechanical thresholds. In contrast, norepinephrine content was significantly increased and correlated with mechanical thresholds in neuropathic, but not sham animals. These results describe changes in striatal signaling in neuropathic pain animals, and contribute to the literature defining the role of dopamine and norepinephrine in mediating sensory thresholds in healthy and neuropathic pain states.
PMCID: PMC4136541  PMID: 25052072
basal ganglia; catecholamines; chronic pain; neuralgia; pain thresholds
2.  Extracellular signal-regulated kinase activation in the amygdala mediates elevated plus maze behavior during opioid withdrawal 
Behavioural pharmacology  2009;20(7):576-583.
This study examined whether activation of extracellular signal-regulated kinase (ERK) contributes to the increased open-arm time observed in the elevated plus maze (EPM) during opioid withdrawal. We applied SL327, a selective ERK kinase (MEK) inhibitor, to specific limbic areas and examined the effect on EPM behaviors of controls and during naloxone-precipitated morphine withdrawal. We next confirmed that ERK activation increased in limbic areas of mice undergoing naloxone-precipitated morphine withdrawal. Direct injection of SL327 into the amygdala blocked the withdrawal-induced increase in open-arm time; however, injecting SL327 into the septum had no effect. Consistent with these results, both 0.2 and 2 mg/kg naloxone increased ERK activation in the central amygdala of morphine-dependent mice. In drug-naïve mice, 2 mg/kg naloxone, but not 0.2 mg/kg, increased ERK activation in the central amygdala. During withdrawal, increased ERK activation was also observed in the lateral septum. In the LC, a significant increase was only observed in morphine-dependent mice receiving 2 mg/kg, but not 0.2 mg/kg, naloxone. It is concluded that ERK activation in limbic areas is likely involved in both the aversive properties of naloxone and in the affective/emotional symptoms of opioid withdrawal, including mediating EPM behaviors.
PMCID: PMC4494789  PMID: 19738463
Morphine; Naloxone; Dependence; Mitogen-activated protein kinase (MAPK); Opioid signaling; mouse
3.  Niclosamide inhibits androgen receptor variants expression and overcomes enzalutamide resistance in castration resistant prostate cancer 
Enzalutamide, a second-generation antiandrogen, was recently approved for the treatment of castration-resistant prostate cancer (CRPC) in patients who no longer respond to docetaxel. Despite these advances that provide temporary respite, resistance to enzalutamide occurs frequently. AR splice variants such as AR-V7 have recently been shown to drive castration resistant growth and resistance to enzalutamide. This study was designed to identify inhibitors of AR variants and test its ability to overcome resistance to enzalutamide.
Experimental Design
The drug screening was conducted using luciferase activity assay to determine the activity of AR-V7 after treatment with the compounds in the Prestwick Chemical Library, which contains about 1120 FDA-approved drugs. The effects of the identified inhibitors on AR-V7 activity and enzalutamide sensitivity were characterized in CRPC and enzalutamide-resistant prostate cancer cells in vitro and in vivo.
Niclosamide, an FDA-approved anti-helminthic drug, was identified as a potent AR-V7 inhibitor in prostate cancer cells. Niclosamide significantly downregulated AR-V7 protein expression by protein degradation through a proteasome dependent pathway. Niclosamide also inhibited AR-V7 transcription activity and reduced the recruitment of AR-V7 to the PSA promoter. Niclosamide inhibited prostate cancer cell growth in vitro and tumor growth in vivo. Furthermore, the combination of niclosamide and enzalutamide resulted in significantly inhibition of enzalutamide-resistant tumor growth, suggesting that Niclosamide enhances enzalutamide therapy and overcomes enzalutamide resistance in castration resistant prostate cancer cells.
Niclosamide was identified as a novel inhibitor of AR variants. Our findings offer preclinical validation of niclosamide as a promising inhibitor of androgen receptor variants to treat, either alone or in combination with current antiandrogen therapies, advanced prostate cancer patients, especially those resistant to enzalutamide.
PMCID: PMC4058390  PMID: 24740322
prostate cancer; enzalutamide; niclosamide; variants; drug resistance
4.  Microglia Disrupt Mesolimbic Reward Circuitry in Chronic Pain 
The Journal of Neuroscience  2015;35(22):8442-8450.
Chronic pain attenuates midbrain dopamine (DA) transmission, as evidenced by a decrease in opioid-evoked DA release in the ventral striatum, suggesting that the occurrence of chronic pain impairs reward-related behaviors. However, mechanisms by which pain modifies DA transmission remain elusive. Using in vivo microdialysis and microinjection of drugs into the mesolimbic DA system, we demonstrate in mice and rats that microglial activation in the VTA compromises not only opioid-evoked release of DA, but also other DA-stimulating drugs, such as cocaine. Our data show that loss of stimulated extracellular DA is due to impaired chloride homeostasis in midbrain GABAergic interneurons. Treatment with minocycline or interfering with BDNF signaling restored chloride transport within these neurons and recovered DA-dependent reward behavior. Our findings demonstrate that a peripheral nerve injury causes activated microglia within reward circuitry that result in disruption of dopaminergic signaling and reward behavior. These results have broad implications that are not restricted to the problem of pain, but are also relevant to affective disorders associated with disruption of reward circuitry. Because chronic pain causes glial activation in areas of the CNS important for mood and affect, our findings may translate to other disorders, including anxiety and depression, that demonstrate high comorbidity with chronic pain.
PMCID: PMC4452552  PMID: 26041913
addiction; affective disorder; chronic pain; dopamine; emotion; opioids
6.  Inhibition of constitutively active Stat3 reverses enzalutamide resistance in LNCaP derivative prostate cancer cells 
The Prostate  2013;74(2):201-209.
Use of enzalutamide has produced a revolutionary change in the treatment of advanced prostate cancer. However, clinical resistance to enzalutamide can develop swiftly in initial responders. This study aimed to test whether overexpression of IL-6 and constitutive activation of Stat3 in prostate cancer cells increase resistance to enzalutamide.
Experimental design
Sensitivity of prostate cancer cells to enzalutamide was tested using cell growth assays and clonogenic assays. Quantitative reverse transcription-PCR, ELISA and Western blotting were performed to detect expression levels of IL-6, c-Myc, survivin and AR. Expression of Stat3 was downregulated using siRNA specific to Stat3. ChIP assay was performed to examine recruitment of AR to the PSA promoter.
Prostate cancer cells expressing autocrine IL-6 are resistant to enzalutamide and autocrine IL-6 leads to constitutive activation of Stat3 and its target genes. Down regulation of Stat3 led to an increase in sensitivity of prostate cancer cells to enzalutamide. Overexpression of constitutively active Stat3 in prostate cancer cells induced resistance to enzalutamide treatment. Constitutively active Stat3 also enhanced the recruitment of AR to PSA promoter which could not be disrupted by enzalutamide. The Stat3 inhibitor AG490 reversed enzalutamide resistance in prostate cancer cells, while combination treatment with enzalutamide and AG490 significantly inhibited cell growth and induced cell apoptosis.
This study demonstrates that the autocrine IL-6 pathway induces enzalutamide resistance in prostate cancer cells via the constitutive activation of Stat3. Co-targeting IL6-Stat3 pathway with enzalutamide may be utilized for treatment of advanced prostate cancer.
PMCID: PMC4437226  PMID: 24307657
Prostate cancer; Interleukin-6; Enzalutamide; Stat3
7.  The role of endogenous PACAP in motor stimulation and conditioned place preference induced by morphine in mice 
Psychopharmacology  2009;204(3):457-463.
The neuropeptide pituitary adenylyl cyclase-activating peptide (PACAP) and its receptors (PAC1 and VPAC2) are expressed in the ventral tegmental area and nucleus accumbens, raising the possibility that PACAP could be a potential modulator of the mesolimbic dopaminergic system.
The present study was designed to determine if PACAP plays a role in acute motor stimulatory and rewarding actions of morphine.
The effect of intracerebroventricular PACAP administration (0, 0.03, 0.3, 1.0, or 3.0 μg/3 μL) was studied on basal motor activity as well as on morphine (5 mg/kg)-stimulated motor activity. Motor stimulation and conditioned place preference (CPP) induced by morphine (5 or 10 mg/kg) were also determined in mice lacking PACAP and their wild-type controls.
Intracerebroventricular PACAP dose-dependently suppressed basal motor activity and PACAP-deficient mice exhibited higher basal motor activity than control mice, providing evidence that the action of endogenous PACAP on basal motor activity is inhibitory. Paradoxically, low doses of PACAP which did not alter basal motor activity were found to enhance the motor stimulatory action of morphine. Furthermore, morphine-induced motor stimulation was blunted in PACAP-deficient mice. Additionally, morphine-induced CPP following a single, but not repeated, alternate-day saline/morphine (10 mg/kg) conditioning was blunted in PACAP-deficient mice compared to their wild-type littermates/controls.
The present results suggest that endogenous PACAP, at low doses, positively modulates the acute motor stimulatory and rewarding actions of morphine.
PMCID: PMC4429892  PMID: 19199096
Pituitary adenylyl cyclase-activating peptide (PACAP); Morphine; Motor activity; Conditioned place preference; Reward; Intracerebroventricular administration; PACAP-deficient mice
8.  Localization and Regulation of Fluorescence-Labeled Delta Opioid Receptor, Expressed in Enteric Neurons of Mice 
Gastroenterology  2011;141(3):982-991.e1-8.
Background & Aims
Opioids and opiates inhibit gastrointestinal functions via μ, δ, and κ receptors. Although agonists of the δ opioid receptor (DOR) suppress motility and secretion, little is known about the localization and regulation of DOR in the gastrointestinal tract.
We studied mice in which the gene that encodes the enhanced green fluorescent protein (eGFP) was inserted into Oprd1, which encodes DOR, to express an ~80 kDa product (DOReGFP). We used these mice to examine how agonists of DOR regulate the subcellular distribution of the DOR.
DOReGFP was expressed in all regions but confined to enteric neurons and fibers within the muscularis externa. In the submucosal plexus, DOReGFP was detected in neuropeptide Y-positive secretomotor and vasodilator neurons of the small intestine, but was rarely observed in the large bowel. In the myenteric plexus of the small intestine, DOReGFP was present in similar proportions of excitatory motoneurons and interneurons that expressed choline acetyltransferase and substance P, and in inhibitory motoneurons and interneurons that contained nitric oxide synthase. DOReGFP was mostly present in nitrergic myenteric neurons of colon. DOReGFP and μ opioid receptors were often co-expressed. DOReGFP-expressing neurons were associated with enkephalin-containing varicosities and enkephalin-induced, clathrin- and dynamin-mediated endocytosis and lysosomal trafficking of DOReGFP. DOReGFP replenishment at the plasma membrane was slow, requiring de novo synthesis, rather than recycling.
DOR localizes specifically to submucosal and myenteric neurons, which might account for the ability of DOR agonists to inhibit gastrointestinal secretion and motility. Sustained down-regulation of DOReGFP at the plasma membrane of activated could induce long-lasting tolerance to DOR agonists.
PMCID: PMC4429902  PMID: 21699782
Trafficking; opiate drug; constipation; morphine; transgenic mice
9.  Autophagy as a Modulator and Target in Prostate Cancer 
Nature reviews. Urology  2014;11(9):508-516.
Autophagy, or “self eating,” is an adaptive process that helps cells cope with metabolic, toxic, and even infectious stressors. While the adaptive capability of autophagy is generally beneficial, autophagy can also facilitate enhanced nutrient utilization and improved growth characteristics in cancer cells. Moreover, autophagy can promote greater cellular robustness in the context of therapeutic intervention. This has proven to be the case in advanced prostate cancer, where preclinical data largely supports that autophagy facilitates both disease progression and therapeutic resistance. Notably, androgen deprivation therapy, taxane-based chemotherapy, targeted kinase inhibition, and nutrient restriction all induce significant cellular distress. Autophagy is subsequently up-regulated through core metabolic regulatory signaling cascades (i.e. AMPK, PI3K, and mTOR), and more favorable growth and nutrient conditions are established. Current research also demonstrates that when the autophagic machinery is inhibited, greater cell killing and tumor responsiveness can be obtained. In this review, we will cover current prostate cancer treatments associated with alterations in autophagy; data supporting autophagic modulation with added emphasis on alterations occurring within prostate cancer models; and finally, research supporting adjuvant autophagic modulation with current prostate cancer treatment paradigms.
PMCID: PMC4415606  PMID: 25134829
10.  Application of Structural Rigidity Analysis to Assess Fidelity of Healed Fractures in Rat Femurs with Critical Defects 
Calcified tissue international  2010;86(5):397-403.
Approximately 6 million fractures occur each year in the United States, with an estimated medical and loss of productivity cost of $99 billion. As our population ages, it can only be expected that these numbers will continue to rise. While there have been recent advances in available treatments for fractures, assessment of the healing process remains a subjective process. This study aims to demonstrate the use of micro-computed tomography (μCT)-based structural rigidity analysis to accurately and quantitatively assess the progression of fracture healing over time in a rat model. The femora of rats with simulated lytic defects were injected with human BMP-2 cDNA at various time points postinjury (t = 0, 1, 5, 10 days) to accelerate fracture healing, harvested 56 days from time of injury, and subjected to μCT imaging to obtain cross-sectional data that were used to compute torsional rigidity. The specimens then underwent torsional testing to failure using a previously described pure torsional testing system. Strong correlations were found between measured torsional rigidity and computed torsional rigidity as calculated from both average (R2 = 0.63) and minimum (R2 = 0.81) structural rigidity data. While both methods were well correlated across the entire data range, minimum torsional rigidity was a better descriptor of bone strength, as seen by a higher Pearson coefficient and smaller y-intercept. These findings suggest considerable promise in the use of structural rigidity analysis of μCT data to accurately and quantitatively measure fracture-healing progression.
PMCID: PMC4405879  PMID: 20354683
Fracture healing; Healing strength; Structural rigidity analysis; Segmental defect; Rat model
11.  Progress in intra-articular therapy 
Nature reviews. Rheumatology  2013;10(1):11-22.
Diarthrodial joints are well suited to intra-articular injection, and the local delivery of therapeutics in this fashion brings several potential advantages to the treatment of a wide range of arthropathies. Possible benefits include increased bioavailability, reduced systemic exposure, fewer adverse events, and lower total drug costs. Nevertheless, intra-articular therapy is challenging because of the rapid egress of injected materials from the joint space; this elimination is true of both small molecules, which exit via synovial capillaries, and of macromolecules, which are cleared by the lymphatic system. In general, soluble materials have an intra-articular dwell time measured only in hours. Corticosteroids and hyaluronate preparations constitute the mainstay of FDA-approved intra-articular therapeutics. Recombinant proteins, autologous blood products and analgesics have also found clinical use via intra-articular delivery. Several alternative approaches, such as local delivery of cell and gene therapy, as well as the use of microparticles, liposomes, and modified drugs, are in various stages of preclinical development.
PMCID: PMC4402210  PMID: 24189839
12.  Altered Relative Expression of BMPs and BMP Inhibitors in Cartilaginous Areas of Human Fractures Progressing towards Nonunion 
The present study was conducted to evaluate the hypothesis that an imbalance in the local production of bone morphogenetic proteins (BMPs) and BMP inhibitors exists within the cartilaginous intermediate of nonhealing fractures. Biopsies were recovered intraoperatively from human fractures that, upon follow-up, were found to heal normally or become nonunions. The samples were examined by immunohistochemistry to determine the expression of BMP-2, BMP-14, and the BMP inhibitors noggin and chordin. Expression was determined semiquantitatively based on the area of positive staining per area of cartilage and by determining the number of positively staining cells and the intensity of staining. There was a significant reduction in BMP-2 and BMP-14 expression in cartilaginous areas of nonhealing fractures compared to healing fractures. However, there was no difference in the expression of the BMP inhibitors between the two groups of fractures. This imbalance in the expression of BMPs and BMP inhibitors within cartilaginous areas of developing nonunions may account for their reduced bone forming ability. These data suggest strategies for preventing the development of nonunions by altering levels of BMPs and their inhibitors within fracture sites.
PMCID: PMC4382003  PMID: 19058174
Fracture; nonunion; BMP; noggin; chordin; cartilage
13.  miR-30 as a tumor suppressor connects EGF/Src signal to ERG and EMT 
Oncogene  2013;33(19):2495-2503.
Src tyrosine kinase (Src) is implicated in the development of bone metastasis and castration-resistance of prostate cancer. Src inhibitors are currently being tested in clinical trials for such diseases. Understanding the molecular and cellular actions of Src inhibitors holds the key to future improvement of this line of therapy. Here we describe the microRNA (miRNA) expression profiles modulated by two Src inhibitors and demonstrate that the miR-30 family members are the most prominently induced species. Consistent with its tumor suppressor role, miR-30 is downmodulated by oncogenic signals such as epidermal growth factor (EGF) and hepatocyte growth factor (HGF), and is generally underexpressed in prostate cancer specimens. A number of epithelial to mesenchymal transition (EMT)-associated genes are predicted targets of miR-30. Among these genes the Ets Related Gene (ERG) is the most frequently overexpressed-oncogene in prostate cancer activated by genomic fusion events between promoter upstream sequences of the TMPRSS2 and coding sequences of ERG. We showed by ERG 3′UTR-reporter and mutagenesis assays that ERG is a direct target of miR-30. Overexpression of miR-30 in prostate cancer cells suppresses EMT phenotypes and inhibits cell migration and invasion. It also inhibits the in vitro and in vivo growth of VCaP cells, which depends on TMPRSS2-ERG for proliferation. TMPRSS2-ERG is generally regulated by androgen at the transcriptional level. Our finding reveals a new post-transcriptional mechanism of TMPRSS2-ERG regulation by Src and growth signals via miR-30 providing a rationale for targeting ERG positive castration resistant tumors with Src inhibitors.
PMCID: PMC4370229  PMID: 23728339
Prostate cancer; EGF; Src; miRNA; ERG and EMT
14.  Perspectives on the Use of Gene Therapy for Chronic Joint Diseases 
Current gene therapy  2008;8(4):273-286.
Advances in molecular and cellular biology have identified a wide variety of proteins including targeted cytokine inhibitors, immunomodulatory proteins, cytotoxic mediators, angiogenesis inhibitors, and intracellular signalling molecules that could be of great benefit in the treatment of chronic joint diseases, such as osteo- and rheumatoid arthritis. Unfortunately, protein-based drugs are difficult to administer effectively. They have a high rate of turnover, requiring frequent readministration, and exposure in non-diseased tissue can lead to serious side effects. Gene transfer technologies offer methods to enhance the efficacy of protein-based therapies, enabling the body to produce these molecules locally at elevated levels for extended periods. The proof of concept of gene therapies for arthritis has been exhaustively demonstrated in multiple laboratories and in numerous animal models. This review attempts to condense these studies and to discuss the relative benefits and limitations of the methods proposed and to discuss the challenges toward translating these technologies into clinical realities.
PMCID: PMC4350777  PMID: 18691023
Arthritis; gene therapy; adenovirus; adeno-associated virus; lentivirus; osteoarthritis; interleukin-1; tumor necrosis factor
European cells & materials  2012;23:94-102.
Tendon rupture is a common injury. Inadequate endogenous repair often leaves patients symptomatic, with tendons susceptible to re-rupture. Administration of certain growth factors improves tendon healing in animal models, but their delivery remains a challenge. Here we evaluated the delivery of TGF-β1 to tendon defects by the implantation of genetically modified muscle grafts. Rat muscle biopsies were transduced with recombinant adenovirus encoding TGF-β1 and grafted onto surgically transected Achilles tendons in recipient animals. Tissue regenerates were compared to those of controls by biomechanical testing as well as histochemical and immunohistochemical analyses. Healing was greatly accelerated when genetically modified grafts were implanted into tendon defects, with the resulting repair tissue gaining nearly normal histological appearance as early as 2 weeks postoperatively. This was associated with decreased deposition of type III collagen in favour of large fibre bundles indicative of type I collagen. These differences in tendon composition coincided with accelerated restoration of mechanical strength. Tendon thickness increased in gene-treated animals at weeks 1 and 2, but by week 8 became significantly lower than that of controls suggesting accelerated remodelling. Thus localised TGF-β1 delivery via adenovirus-modified muscle grafts improved tendon healing in this rat model and holds promise for clinical application.
PMCID: PMC4339190  PMID: 22354460
Transforming growth factor-β; tendon healing; gene transfer; muscle graft; adenovirus
16.  Gene therapy for bone healing 
Clinical problems in bone healing include large segmental defects, nonunion and delayed union of fractures, and spinal fusions. Gene-transfer technologies have the potential to aid healing by permitting the local delivery and sustained expression of osteogenic gene products within osseous lesions. Key questions for such an approach include the choice of transgene, vector and gene-transfer strategy. Most experimental data have been obtained using cDNAs encoding osteogenic growth factors such as bone morphogenetic protein-2 (BMP-2), BMP-4 and BMP-7, in conjunction with both nonviral and viral vectors using in vivo and ex vivo delivery strategies. Proof of principle has been convincingly demonstrated in small-animal models. Relatively few studies have used large animals, but the results so far are encouraging. Once a reliable method has been developed, it will be necessary to perform detailed pharmacological and toxicological studies, as well as satisfy other demands of the regulatory bodies, before human clinical trials can be initiated. Such studies are very expensive and often protracted. Thus, progress in developing a clinically useful gene therapy for bone healing is determined not only by scientific considerations, but also by financial constraints and the ambient regulatory environment.
PMCID: PMC4339214  PMID: 20569532
18.  Does the kappa opioid receptor system contribute to pain aversion? 
The kappa opioid receptor (KOR) and the endogenous peptide-ligand dynorphin have received significant attention due the involvement in mediating a variety of behavioral and neurophysiological responses, including opposing the rewarding properties of drugs of abuse including opioids. Accumulating evidence indicates this system is involved in regulating states of motivation and emotion. Acute activation of the KOR produces an increase in motivational behavior to escape a threat, however, KOR activation associated with chronic stress leads to the expression of symptoms indicative of mood disorders. It is well accepted that KOR can produce analgesia and is engaged in chronic pain states including neuropathic pain. Spinal studies have revealed KOR-induced analgesia in reversing pain hypersensitivities associated with peripheral nerve injury. While systemic administration of KOR agonists attenuates nociceptive sensory transmission, this effect appears to be a stress-induced effect as anxiolytic agents, including delta opioid receptor agonists, mitigate KOR agonist-induced analgesia. Additionally, while the role of KOR and dynorphin in driving the dysphoric and aversive components of stress and drug withdrawal has been well characterized, how this system mediates the negative emotional states associated with chronic pain is relatively unexplored. This review provides evidence that dynorphin and the KOR system contribute to the negative affective component of pain and that this receptor system likely contributes to the high comorbidity of mood disorders associated with chronic neuropathic pain.
PMCID: PMC4233910  PMID: 25452729
kappa opioid receptor; pain; aversion; reward system ventral tegmental area; dopamine; negative reinforcement
19.  Opioid self-administration results in cell-type specific adaptations of striatal medium spiny neurons 
Behavioural brain research  2013;256:10.1016/j.bbr.2013.08.009.
Medium-sized spiny neurons (MSNs), the predominant neuronal population of the striatum, are an integral component of the many cortical and limbic pathways associated with reward-related behaviors. A differential role of the D1 receptor-enriched (D1) MSNs of the striatonigral direct pathway, as compared with the D2 receptor-enriched (D2) MSNs of the striatopallidal indirect pathway, in mediating the addictive behaviors associated with cocaine is beginning to emerge. However, whether opioids, well-known analgesics with euphoric properties, similarly induce dissociable signaling adaptations in these neurons remains unclear. Transgenic mice expressing Green Fluorescent Protein (GFP)-labeled D1 or D2 neurons were implanted with intravenous jugular catheters and trained to self-administer the opioid remifentanil. D1- and D2-GFP mice learned to self-administer 0.1 mg/kg/infusion remifentanil during 2hr sessions over 13 contiguous days. Thereafter, the electrophysiological properties of D1 and D2 MSNs in the shell region of the nucleus accumbens (NAc) were assessed. We found that prior opioid exposure did not alter the basic membrane properties nor the kinetics or amplitude of miniature excitatory postsynaptic currents (mEPSCs). However, when challenged with the mu opioid receptor (µOR) agonist DAMGO, the characteristic inhibitory profile of this receptor was altered. DAMGO inhibited the frequency of mEPSCs in D1-MSNs from control mice receiving saline and in D2-MSNs from mice exposed to remifentanil or saline, but this inhibitory profile was reduced in D1-MSNs from mice receiving remifentanil. Remifentanil exposure also altered the probability of glutamate release onto D1-, but not D2-MSNs. Together these results suggest a D1-pathway specific effect associated with the acquisition of opioid-seeking behaviors.
PMCID: PMC3815993  PMID: 23968589
striatum; medium spiny neurons; intravenous self-administration; mu opioid receptor; electrophysiology; mice
20.  Advances in Regenerative Orthopaedics 
Mayo Clinic proceedings  2013;88(11):1323-1339.
Orthopaedic injuries are very common and a source of much misery and economic stress. Several relevant tissues, such as cartilage, meniscus and intra-articular ligaments, do not heal. And even bone, which normally regenerates spontaneously, can fail to mend. The regeneration of orthopaedic tissues requires four key components: cells, morphogenetic signals, scaffolds and an appropriate mechanical environment. Although differentiated cells from the tissue in question can be used, most cellular research focuses on the use mesenchymal stem cells (MSCs). These can be retrieved from many different tissues, and one unresolved question is the degree to which the origin of the cells matters. Embryonic and induced, pluripotential stem cells are also under investigation. Morphogenetic signals are most frequently supplied by individual, recombinant growth factors or native mixtures provided by, for instance, platelet-rich plasma; MSCs are also a rich source of trophic factors. Obstacles to the sustained delivery of individual growth factors can be addressed by gene transfer or smart scaffolds, but we still lack detailed, necessary information on which delivery profiles are needed. Scaffolds may be based upon natural products, synthetic materials, or devitalized extracellular matrix. Strategies to combine these components to regenerate tissue can follow traditional tissue engineering practices, but these are costly, cumbersome and not well suited to treating large numbers of individuals. More expeditious approaches make full use of intrinsic biological processes in vivo to avoid the need for ex vivo expansion of autologous cells and multiple procedures. Clinical translation remains a bottleneck.
PMCID: PMC4214280  PMID: 24182709
21.  Preclinical toxicity evaluation of AAV for pain: evidence from human AAV studies and from the pharmacology of analgesic drugs 
Molecular Pain  2014;10:54.
Gene therapy with adeno-associated virus (AAV) has advanced in the last few years from promising results in animal models to >100 clinical trials (reported or under way). While vector availability was a substantial hurdle a decade ago, innovative new production methods now routinely match the scale of AAV doses required for clinical testing. These advances may become relevant to translational research in the chronic pain field. AAV for pain targeting the peripheral nervous system was proven to be efficacious in rodent models several years ago, but has not yet been tested in humans. The present review addresses the steps needed for translation of AAV for pain from the bench to the bedside focusing on pre-clinical toxicology. We break the potential toxicities into three conceptual categories of risk: First, risks related to the delivery procedure used to administer the vector. Second, risks related to AAV biology, i.e., effects of the vector itself that may occur independently of the transgene. Third, risks related to the effects of the therapeutic transgene. To identify potential toxicities, we consulted the existing evidence from AAV gene therapy for other nervous system disorders (animal toxicology and human studies) and from the clinical pharmacology of conventional analgesic drugs. Thereby, we identified required preclinical studies and charted a hypothetical path towards a future phase I/II clinical trial in the oncology-palliative care setting.
PMCID: PMC4237902  PMID: 25183392
Adeno-associated virus; Pain; Gene therapy; Toxicology; Interleukin-10; Beta-endorphin
22.  Inhibition of ABCB1 expression overcomes acquired docetaxel resistance in prostate cancer 
Molecular cancer therapeutics  2013;12(9):1829-1836.
Docetaxel is the first-line standard treatment for castration resistant prostate cancer (CRPC). However, relapse eventually occurs due to the development of resistance to docetaxel. In order to unravel the mechanism of acquired docetaxel resistance, we established docetaxel-resistant prostate cancer cells, TaxR, from castration resistant C4-2B prostate cancer cells. The IC50 for docetaxel in TaxR cells was about 70-fold higher than parental C4-2B cells. Global gene expression analysis revealed alteration of expression of a total of 1604 genes with 52% being upregulated and 48% downregulated. ABCB1, which belongs to the ATP-binding cassette (ABC) transporter family, was identified among the top upregulated genes in TaxR cells. The role of ABCB1 in the development of docetaxel resistance was examined. Knockdown of ABCB1 expression by its specific shRNA or inhibitor resensitized docetaxel resistant TaxR cells to docetaxel treatment by enhancing apoptotic cell death. Furthermore, we identified that apigenin, a natural product of the flavone family, inhibits ABCB1 expression and resensitizes docetaxel resistant prostate cancer cells to docetaxel treatment. Collectively, these results suggest that overexpression of ABCB1 mediates acquired docetaxel resistance and targeting ABCB1 expression could be potential approach to resensitize docetaxel resistant prostate cancer cells to docetaxel treatment.
PMCID: PMC3947549  PMID: 23861346
docetaxel; prostate cancer; ABCB1
23.  Molecular pathways and targets in prostate cancer 
Oncotarget  2014;5(17):7217-7259.
Prostate cancer co-opts a unique set of cellular pathways in its initiation and progression. The heterogeneity of prostate cancers is evident at earlier stages, and has led to rigorous efforts to stratify the localized prostate cancers, so that progression to advanced stages could be predicted based upon salient features of the early disease. The deregulated androgen receptor signaling is undeniably most important in the progression of the majority of prostate tumors. It is perhaps because of the primacy of the androgen receptor governed transcriptional program in prostate epithelium cells that once this program is corrupted, the consequences of the ensuing changes in activity are pleotropic and could contribute to malignancy in multiple ways. Following localized surgical and radiation therapies, 20-40% of patients will relapse and progress, and will be treated with androgen deprivation therapies. The successful development of the new agents that inhibit androgen signaling has changed the progression free survival in hormone resistant disease, but this has not changed the almost ubiquitous development of truly resistant phenotypes in advanced prostate cancer. This review summarizes the current understanding of the molecular pathways involved in localized and metastatic prostate cancer, with an emphasis on the clinical implications of the new knowledge.
PMCID: PMC4202120  PMID: 25277175
prostate cancer; molecular targets; CRPC; localized prostate cancer
24.  Targeted Expression of Mu-Opioid Receptors in a Subset of Striatal Direct-Pathway Neurons Restores Opiate Reward 
Nature neuroscience  2014;17(2):254-261.
Mu-Opioid Receptors (MOR) are necessary for the analgesic and addictive effects of opioids such as morphine, but the MOR-expressing neuronal populations that mediate the distinct opiate effects remain elusive. Here we devised a novel conditional BAC rescue strategy to show that mice with targeted MOR expression in a subpopulation of striatal direct-pathway neurons enriched in the striosome and nucleus accumbens, in an otherwise MOR-null background, restore opiate reward, opiate-induced striatal dopamine release, and partially restore motivation to self-administer opiates. However, they lack opiate analgesia or withdrawal. Importantly, we used Cre-mediated deletion of the rescued MOR transgene to establish that striatal, rather than a few extrastriatal sites of MOR transgene expression, is needed for the restoration of opiate reward. Together, our study demonstrates that a subpopulation of striatal direct-pathway neurons is sufficient to support opiate reward-driven behaviors and provides a novel intersectional genetic approach to dissect neurocircuit-specific gene function in vivo.
PMCID: PMC4008330  PMID: 24413699
25.  Muc5b Is Required for Airway Defense 
Nature  2013;505(7483):412-416.
Respiratory surfaces are exposed to billions of particulates and pathogens daily. A protective mucus barrier traps and eliminates them via mucociliary clearance (MCC)1,2. However, excessive mucus contributes to transient respiratory infections and to the pathogenesis of numerous respiratory diseases1. MUC5AC and MUC5B are evolutionarily conserved genes that encode structurally related mucin glycoproteins, the principal macromolecules in airway mucus1,3. Genetic variants are linked to diverse lung diseases4-6, but specific roles for MUC5AC and MUC5B in MCC, and the lasting effects of their inhibition, are unknown. Here we show that Muc5b (but not Muc5ac) is required for MCC, for controlling infections in the airways and middle ear, and for maintaining immune homeostasis in the lungs. Muc5b deficiency caused materials to accumulate in upper and lower airways. This defect led to chronic infection by multiple bacterial species, including Staphylococcus aureus, and to inflammation that failed to resolve normally7. Apoptotic macrophages accumulated, phagocytosis was impaired, and IL-23 production was reduced inMuc5b−/− mice. By contrast, in Muc5b transgenic (Tg) mice, macrophage functions improved. Existing dogma defines mucous phenotypes in asthma and chronic obstructive pulmonary disease (COPD) as driven by increased MUC5AC, with MUC5B levels either unaffected or increased in expectorated sputum1,8. However, in many patients, MUC5B production at airway surfaces decreases by as much as 90%9-11. By distinguishing a specific role for Muc5b in MCC, and by determining its impact on bacterial infections and inflammation in mice, our results provide a refined framework for designing targeted therapies to control mucin secretion and restore MCC.
PMCID: PMC4001806  PMID: 24317696

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