Regulation of Neuromodulator Receptor Efficacy - Implications for Whole-Neuron and Synaptic Plasticity

Membrane receptors for neuromodulators (NM) are highly regulated in their distribution and efficacy - a phenomenon which influences the individual cell's response to central signals of NM release. Even though NM receptor regulation is implicated in the pharmacological action of many drugs, and is also known to be influenced by various environmental factors, its functional consequences and modes of action are not well understood. In this paper we summarize relevant experimental evidence on NM receptor regulation (specifically dopamine D1 and D2 receptors) in order to explore its significance for neural and synaptic plasticity. We identify the relevant components of NM receptor regulation (receptor phosphorylation, receptor trafficking and sensitization of second-messenger pathways) gained from studies on cultured cells. Key principles in the regulation and control of short-term plasticity (sensitization) are identified, and a model is presented which employs direct and indirect feedback regulation of receptor efficacy. We also discuss long-term plasticity which involves shifts in receptor sensitivity and loss of responsivity to NM signals. Finally, we discuss the implications of NM receptor regulation for models of brain plasticity and memorization. We emphasize that a realistic model of brain plasticity will have to go beyond Hebbian models of long-term potentiation and depression. Plasticity in the distribution and efficacy of NM receptors may provide another important source of functional plasticity with implications for learning and memory.Comment: 35 page

A single extracellular amino acid in Free Fatty Acid Receptor 2 defines antagonist species selectivity and G protein selection bias

Free Fatty Acid Receptor 2 is a GPCR activated by short chain fatty acids produced in high levels in the lower gut by microbial fermentation of non-digestible carbohydrates. A major challenge in studying this receptor is that the mouse ortholog does not have significant affinity for antagonists that are able to block the human receptor. Docking of exemplar antagonists from two chemical series to homology models of both human and mouse Free Fatty Acid Receptor 2 suggested that a single lysine - arginine variation at the extracellular face of the receptor might provide the basis for antagonist selectivity and mutational swap studies confirmed this hypothesis. Extending these studies to agonist function indicated that although the lysine - arginine variation between human and mouse orthologs had limited effect on G protein-mediated signal transduction, removal of positive charge from this residue produced a signalling-biased variant of Free Fatty Acid Receptor 2 in which Gi-mediated signalling by both short chain fatty acids and synthetic agonists was maintained whilst there was marked loss of agonist potency for signalling via Gq/11 and G12/13 G proteins. A single residue at the extracellular face of the receptor thus plays key roles in both agonist and antagonist function

Ligand-guided homology modeling drives identification of novel histamine H3 receptor ligands

In this study, we report a ligand-guided homology modeling approach allowing the analysis of relevant binding site residue conformations and the identification of two novel histamine H3 receptor ligands with binding affinity in the nanomolar range. The newly developed method is based on exploiting an essential charge interaction characteristic for aminergic G-protein coupled receptors for ranking 3D receptor models appropriate for the discovery of novel compounds through virtual screening

MET Gene Amplification and MET Receptor Activation Are Not Sufficient to Predict Efficacy of Combined MET and EGFR Inhibitors in EGFR TKI-Resistant NSCLC Cells

Epidermal growth factor receptor (EGFR), member of the human epidermal growth factor receptor (HER) family, plays a critical role in regulating multiple cellular processes including proliferation, differentiation, cell migration and cell survival. Deregulation of the EGFR signaling has been found to be associated with the development of a variety of human malignancies including lung, breast, and ovarian cancers, making inhibition of EGFR the most promising molecular targeted therapy developed in the past decade against cancer. Human non small cell lung cancers (NSCLC) with activating mutations in the EGFR gene frequently experience significant tumor regression when treated with EGFR tyrosine kinase inhibitors (TKIs), although acquired resistance invariably develops. Resistance to TKI treatments has been associated to secondary mutations in the EGFR gene or to activation of additional bypass signaling pathways including the ones mediated by receptor tyrosine kinases, Fas receptor and NF-kB. In more than 30-40% of cases, however, the mechanisms underpinning drug-resistance are still unknown. The establishment of cellular and mouse models can facilitate the unveiling of mechanisms leading to drug-resistance and the development or validation of novel therapeutic strategies aimed at overcoming resistance and enhancing outcomes in NSCLC patients. Here we describe the establishment and characterization of EGFR TKI-resistant NSCLC cell lines and a pilot study on the effects of a combined MET and EGFR inhibitors treatment. The characterization of the erlotinib-resistant cell lines confirmed the association of EGFR TKI resistance with loss of EGFR gene amplification and/or AXL overexpression and/or MET gene amplification and MET receptor activation. These cellular models can be instrumental to further investigate the signaling pathways associated to EGFR TKI-resistance. Finally the drugs combination pilot study shows that MET gene amplification and MET receptor activation are not sufficient to predict a positive response of NSCLC cells to a cocktail of MET and EGFR inhibitors and highlights the importance of identifying more reliable biomarkers to predict the efficacy of treatments in NSCLC patients resistant to EGFR TKI

Interactions between the neuromodulatory systems and the amygdala: exploratory survey using the Allen Mouse Brain Atlas.

Neuromodulatory systems originate in nuclei localized in the subcortical region of the brain and control fundamental behaviors by interacting with many areas of the central nervous system. An exploratory survey of the cholinergic, dopaminergic, noradrenergic, and serotonergic receptor expression energy in the amygdala, and in the neuromodulatory areas themselves was undertaken using the Allen Mouse Brain Atlas. The amygdala was chosen because of its importance in cognitive behavior and its bidirectional interaction with the neuromodulatory systems. The gene expression data of 38 neuromodulatory receptor subtypes were examined across 13 brain regions. The substantia innominata of the basal forebrain and regions of the amygdala had the highest amount of receptor expression energy for all four neuromodulatory systems examined. The ventral tegmental area also displayed high receptor expression of all four neuromodulators. In contrast, the locus coeruleus displayed low receptor expression energy overall. In general, cholinergic receptor expression was an order of magnitude greater than other neuromodulatory receptors. Since the nuclei of these neuromodulatory systems are thought to be the source of specific neurotransmitters, the projections from these nuclei to target regions may be inferred by receptor expression energy. The comprehensive analysis revealed many connectivity relations and receptor localization that had not been previously reported. The methodology presented here may be applied to other neural systems with similar characteristics, and to other animal models as these brain atlases become available

A novel inhibitor of p75-neurotrophin receptor improves functional outcomes in two models of traumatic brain injury.

The p75 neurotrophin receptor is important in multiple physiological actions including neuronal survival and neurite outgrowth during development, and after central nervous system injury. We have discovered a novel piperazine-derived compound, EVT901, which interferes with p75 neurotrophin receptor oligomerization through direct interaction with the first cysteine-rich domain of the extracellular region. Using ligand binding assays with cysteine-rich domains-fused p75 neurotrophin receptor, we confirmed that EVT901 interferes with oligomerization of full-length p75 neurotrophin receptor in a dose-dependent manner. Here we report that EVT901 reduces binding of pro-nerve growth factor to p75 neurotrophin receptor, blocks pro-nerve growth factor induced apoptosis in cells expressing p75 neurotrophin receptor, and enhances neurite outgrowth in vitro Furthermore, we demonstrate that EVT901 abrogates p75 neurotrophin receptor signalling by other ligands, such as prion peptide and amyloid-β. To test the efficacy of EVT901 in vivo, we evaluated the outcome in two models of traumatic brain injury. We generated controlled cortical impacts in adult rats. Using unbiased stereological analysis, we found that EVT901 delivered intravenously daily for 1 week after injury, reduced lesion size, protected cortical neurons and oligodendrocytes, and had a positive effect on neurological function. After lateral fluid percussion injury in adult rats, oral treatment with EVT901 reduced neuronal death in the hippocampus and thalamus, reduced long-term cognitive deficits, and reduced the occurrence of post-traumatic seizure activity. Together, these studies provide a new reagent for altering p75 neurotrophin receptor actions after injury and suggest that EVT901 may be useful in treatment of central nervous system trauma and other neurological disorders where p75 neurotrophin receptor signalling is affected

Nothing Besides Remains: Preserving the Scientific and Cultural Value of Paleontological Resources in the United States

Receptor occupancy assessed by Positron Emission Tomography (PET) can provide important translational information to help bridge information from one drug to another or from animal to man. The aim of this thesis was to develop nonlinear mixed effects methods for estimation of the relationship between drug exposure and receptor occupancy for the two mGluR5 antagonists AZD9272 and AZD2066 and for the 5HT1B receptor antagonist AZD3783. Also the optimal design for improved estimation of the relationship between drug exposure and receptor occupancy as well as for improved dose finding in neuropathic pain treatment, was investigated. Different modeling approaches were applied. For AZD9272, the radioligand kinetics and receptor occupancy was simultaneously estimated using arterial concentrations as input function and including two brain regions of interest. For AZD2066, a model was developed where brain/plasma partition coefficients from ten different brain regions were included simultaneously as observations. For AZD3783, the simplified reference tissue model was extended to allow different non-specific binding in the reference region and brain regions of interest and the possibility of using white matter as reference was also evaluated. The optimal dose-selection for improved precision of receptor occupancy as well as for improved precision of the minimum effective dose of a neuropathic pain treatment was assessed, using the D-optimal as well as the Ds-optimal criteria. Simultaneous modelling of radioligand and occupancy provided a means to avoid simplifications or approximations and provided the possibility to tests or to relax assumptions. Inclusion of several brain regions of different receptor density simultaneously in the analysis, markedly improved the precision of the affinity parameter. Higher precision was achieved in relevant parameters with designs based on the Ds compared to the D-optimal criterion. The optimal design for improved precision of the relationship between dose and receptor occupancy depended on the number of brain regions and the receptor density of these regions. In conclusion, this thesis presents novel non-linear mixed effects models estimating the relationship between drug exposure and receptor occupancy, providing useful translational information, allowing for a better informed drug-development

NMR Line Shapes and Multi-State Binding Equilibria

Biological function of proteins relies on conformational transitions and binding of specific ligands. Protein-ligand interactions are thermodynamically and kinetically coupled to conformational changes in protein structures as conceptualized by the models of pre-existing equilibria and induced fit. NMR spectroscopy is particularly sensitive to complex ligand-binding modes—NMR line-shape analysis can provide for thermodynamic and kinetic constants of ligand-binding equilibria with the site-specific resolution. However, broad use of line shape analysis is hampered by complexity of NMR line shapes in multi-state systems. To facilitate interpretation of such spectral patterns, I computationally explored systems where isomerization or dimerization of a protein (receptor) molecule is coupled to binding of a ligand. Through an extensive analysis of multiple exchange regimes for a family of three-state models, I identified signature features to guide an NMR experimentalist in recognizing specific interaction mechanisms. Results also show that distinct multistate models may produce very similar spectral patterns. I also discussed aggregation of a receptor as a possible source of spurious three-state line shapes and provided specific suggestions for complementary experiments that can ensure reliable mechanistic insight

ROR-γ drives androgen receptor expression and represents a therapeutic target in castration-resistant prostate cancer.

The androgen receptor (AR) is overexpressed and hyperactivated in human castration-resistant prostate cancer (CRPC). However, the determinants of AR overexpression in CRPC are poorly defined. Here we show that retinoic acid receptor-related orphan receptor γ (ROR-γ) is overexpressed and amplified in metastatic CRPC tumors, and that ROR-γ drives AR expression in the tumors. ROR-γ recruits nuclear receptor coactivator 1 and 3 (NCOA1 and NCOA3, also known as SRC-1 and SRC-3) to an AR-ROR response element (RORE) to stimulate AR gene transcription. ROR-γ antagonists suppress the expression of both AR and its variant AR-V7 in prostate cancer (PCa) cell lines and tumors. ROR-γ antagonists also markedly diminish genome-wide AR binding, H3K27ac abundance and expression of the AR target gene network. Finally, ROR-γ antagonists suppressed tumor growth in multiple AR-expressing, but not AR-negative, xenograft PCa models, and they effectively sensitized CRPC tumors to enzalutamide, without overt toxicity, in mice. Taken together, these results establish ROR-γ as a key player in CRPC by acting upstream of AR and as a potential therapeutic target for advanced PCa

The atypical chemokine receptor Ackr2 constrains NK cell migratory activity and promotes metastasis

Chemokines have been shown to be essential players in a range of cancer contexts. In this study, we demonstrate that mice deficient in the atypical chemokine receptor Ackr2 display impaired development of metastasis in vivo in both cell line and spontaneous models. Further analysis reveals that this relates to increased expression of the chemokine receptor CCR2, specifically by KLRG1+ NK cells from the Ackr2−/− mice. This leads to increased recruitment of KLRG1+ NK cells to CCL2-expressing tumors and enhanced tumor killing. Together, these data indicate that Ackr2 limits the expression of CCR2 on NK cells and restricts their tumoricidal activity. Our data have important implications for our understanding of the roles for chemokines in the metastatic process and highlight Ackr2 and CCR2 as potentially manipulable therapeutic targets in metastasis