Madhara Udawela

Novel Receptor Partners and Function of Receptor Activity-modifying Proteins

The receptor activity-modifying proteins (RAMPs) comprise a family of three accessory proteins that heterodimerize with the calcitonin receptor-like receptor (CL receptor) or with the calcitonin receptor (CTR) to generate different receptor phenotypes. However, RAMPs are more widely distributed across cell and tissue types than the CTR and CL receptor, suggesting additional roles for RAMPs in cellular processes. We have investigated the potential for RAMP interaction with a number of Class II G protein-coupled receptors (GPCRs) in addition to the CL receptor and the CTR. Using immunofluorescence confocal microscopy, we demonstrate, for the first time, that RAMPs interact with at least four additional receptors, the VPAC1 vasoactive intestinal polypeptide/pituitary adenylate cyclase-activating peptide receptor with all three RAMPs; the glucagon and PTH1 parathyroid hormone receptors with RAMP2; and the PTH2 receptor with RAMP3. Unlike the interaction of RAMPs with the CL receptor or the CTR, VPAC1R-RAMP complexes do not show altered phenotypic behavior compared with the VPAC1R alone, as determined using radioligand binding in COS-7 cells. However, the VPAC1R-RAMP2 heterodimer displays a significant enhancement of agonist-mediated phosphoinositide hydrolysis with no change in cAMP stimulation compared with the VPAC1R alone. Our findings identify a new functional consequence of RAMP-receptor interaction, suggesting that RAMPs play a more general role in modulating cell signaling through other GPCRs than is currently appreciated.

Autophagy has a key role in the pathophysiology of schizophrenia

Autophagy is a process preserving the balance between synthesis, degradation and recycling of cellular components and is therefore essential for neuronal survival and function. Several key proteins govern the autophagy pathway including beclin1 and microtubule associated protein 1 light chain 3 (LC3). Here, we show a brain-specific reduction in beclin1 expression in postmortem hippocampus of schizophrenia patients, not detected in peripheral lymphocytes. This is in contrast with activity-dependent neuroprotective protein (ADNP) and ADNP2, which we have previously found to be deregulated in postmortem hippocampal samples from schizophrenia patients, but that now showed a significantly increased expression in lymphocytes from related patients, similar to increases in the anti-apoptotic, beclin1-interacting, Bcl2. The increase in ADNP was associated with the initial stages of the disease, possibly reflecting a compensatory effect. The increase in ADNP2 might be a consequence of neuroleptic treatment, as seen in rats subjected to clozapine treatment. ADNP haploinsufficiency in mice, which results in age-related neuronal death, cognitive and social dysfunction, exhibited reduced hippocampal beclin1 and increased Bcl2 expression (mimicking schizophrenia and normal human aging). At the protein level, ADNP co-immunoprecipitated with LC3B suggesting a direct association with the autophagy process and paving the path to novel targets for drug design.

Complexing Receptor Pharmacology

Abstract:  The most well‐characterized subgroup of family B G protein–coupledreceptors (GPCRs) comprises receptors for peptide hormones, such as secretin, calcitonin (CT), glucagon, and vasoactive intestinal peptide (VIP). Recent data suggest that many of these receptors can interact with a novel family of GPCR accessory proteins termed receptor activity modifying proteins (RAMPs). RAMP interaction with receptors can lead to a variety of actions that include chaperoning of the receptor protein to the cell surface as is the case for the calcitonin receptor‐like receptor (CLR) and the generation of novel receptor phenotypes. RAMP heterodimerization with the CLR and related CT receptor is required for the formation of specific CT gene‐related peptide, adrenomedullin (AM) or amylin receptors. More recent work has revealed that the specific RAMP present in a heterodimer may modulate other functions such as receptor internalization and recycling and also the strength of activation of downstream signaling pathways. In this article we review our current state of knowledge of the consequence of RAMP interaction with family B GPCRs.

Distinct Receptor Activity-Modifying Protein Domains Differentially Modulate Interaction with Calcitonin Receptors

Calcitonin receptors (CTRs) dimerize with receptor activity-modifying proteins (RAMPs) to generate high-affinity amylin (AMY) receptors; however, the relative contribution of individual RAMP domains to the formation of AMY receptors is poorly understood. We have used chimeras between RAMP1 and RAMP2 that specifically exchanged the N-terminal, transmembrane, or C-terminal domain and examined these in assays of [125I]amylin binding or peptide-induced cAMP signaling in COS-7 cells transiently transfected with wild-type or chimeric RAMPs and human CTRa. The specificity of peptides in competition for [125I]amylin binding was principally dictated by the N-terminal domain present in the chimeras; however, the maximal level of binding induced was dictated by the transmembrane domain present. This extended previous data (Zumpe et al., 2000) to provide a distinction between the transmembrane domain and the C terminus in this function. In contrast to the effects on binding, each of the RAMP domains played a role in the signaling phenotype of the receptors. In particular, the potency of calcitonin gene-related peptide (CGRP) was most influenced by the C-terminal domain present, in which the presence of the RAMP1 C-terminal domain led to increased potency over CTRa alone, whereas chimeras with the RAMP2 C-terminal domain did not induce increased CGRP potency. The data provide additional support for the importance of the N terminus in determining binding affinity but reveal a prominent role of the transmembrane domain in the strength of amylin binding and a unique role for the C terminus in signaling by peptides to stimulate cAMP production.

Cholinergic connectivity: it's implications for psychiatric disorders

Acetylcholine has been implicated in both the pathophysiology and treatment of a number of psychiatric disorders, with most of the data related to its role and therapeutic potential focusing on schizophrenia. However, there is little thought given to the consequences of the documented changes in the cholinergic system and how they may affect the functioning of the brain. This review looks at the cholinergic system and its interactions with the intrinsic neurotransmitters glutamate and gamma-amino butyric acid as well as those with the projection neurotransmitters most implicated in the pathophysiologies of psychiatric disorders; dopamine and serotonin. In addition, with the recent focus on the role of factors normally associated with inflammation in the pathophysiologies of psychiatric disorders, links between the cholinergic system and these factors will also be examined. These interfaces are put into context, primarily for schizophrenia, by looking at the changes in each of these systems in the disorder and exploring, theoretically, whether the changes are interconnected with those seen in the cholinergic system. Thus, this review will provide a comprehensive overview of the connectivity between the cholinergic system and some of the major areas of research into the pathophysiologies of psychiatric disorders, resulting in a critical appraisal of the potential outcomes of a dysregulated central cholinergic system.

A Role for Estrogen in Schizophrenia: Clinical and Preclinical Findings

Gender differences in schizophrenia have been extensively researched and it is being increasingly accepted that gonadal steroids are strongly attributed to this phenomenon. Of the various hormones implicated, the estrogen hypothesis has been the most widely researched one and it postulates that estrogen exerts a protective effect by buffering females against the development and severity of the illness. In this review, we comprehensively analyse studies that have investigated the effects of estrogen, in particular 17β-estradiol, in clinical, animal, and molecular research with relevance to schizophrenia. Specifically, we discuss the current evidence on estrogen dysfunction in schizophrenia patients and review the clinical findings on the use of estradiol as an adjunctive treatment in schizophrenia patients. Preclinical research that has used animal models and molecular probes to investigate estradiols underlying protective mechanisms is also substantially discussed, with particular focus on estradiols impact on the major neurotransmitter systems implicated in schizophrenia, namely, the dopamine, serotonin, and glutamate systems.

Biomarkers in schizophrenia: A focus on blood based diagnostics and theranostics

Identifying biomarkers that can be used as diagnostics or predictors of treatment response (theranostics) in people with schizophrenia (Sz) will be an important step towards being able to provide personalized treatment. Findings from the studies in brain tissue have not yet been translated into biomarkers that are practical in clinical use because brain biopsies are not acceptable and neuroimaging techniques are expensive and the results are inconclusive. Thus, in recent years, there has been search for blood-based biomarkers for Sz as a valid alternative. Although there are some encouraging preliminary data to support the notion of peripheral biomarkers for Sz, it must be acknowledged that Sz is a complex and heterogeneous disorder which needs to be further dissected into subtype using biological based and clinical markers. The scope of this review is to critically examine published blood-based biomarker of Sz, focusing on possible uses for diagnosis, treatment response, or their relationship with schizophrenia-associated phenotype. We sorted the studies into six categories which include: (1) brain-derived neurotrophic factor; (2) inflammation and immune function; (3) neurochemistry; (4) oxidative stress response and metabolism; (5) epigenetics and microRNA; and (6) transcriptome and proteome studies. This review also summarized the molecules which have been conclusively reported as potential blood-based biomarkers for Sz in different blood cell types. Finally, we further discusses the pitfall of current blood-based studies and suggest that a prediction model-based, Sz specific, blood oriented study design as well as standardize blood collection conditions would be useful for Sz biomarker development.

A critical role for the short intracellular C terminus in receptor activity-modifying protein function.

Receptor activity-modifying proteins (RAMPs) interact with and modify the behavior of the calcitonin receptor (CTR) and calcitonin receptor-like receptor (CLR). We have examined the contribution of the short intracellular C terminus, using constructs that delete the last eight amino acids of each RAMP. C-Terminal deletion of individual RAMPs had little effect on the signaling profile induced when complexed with CLR in COS-7 or human embryonic kidney (HEK)293 cells. Likewise, confocal microscopy revealed each of the mutant RAMPs translocated hemagglutinin-tagged CLR to the cell surface. In contrast, a pronounced effect of RAMP C-terminal truncation was seen for RAMP/CTRa complexes, studied in COS-7 cells, with significant attenuation of amylin receptor phenotype induction that was stronger for RAMP1 and -2 than RAMP3. The loss of amylin binding upon C-terminal deletion could be partially recovered with overexpression of Gαs, suggesting an impact of the RAMP C terminus on coupling of G proteins to the receptor complex. In HEK293 cells the c-Myc-RAMP1 C-terminal deletion mutant showed high receptor-independent cell surface expression; however, this construct showed low cell surface expression when expressed alone in COS-7 cells, indicating interaction of RAMPs with other cellular components via the C terminus. This mutant also had reduced cell surface expression when coexpressed with CTR. Thus, this study reveals important functionality of the RAMP C-terminal domain and identifies key differences in the role of the RAMP C terminus for CTR versus CLR-based receptors.

Phospholipase C beta 1 expression in the dorsolateral prefrontal cortex from patients with schizophrenia at different stages of illness

Objective: Our recent microarray study detected decreases in the expression of phospholipase C beta 1 mRNA in the dorsolateral prefrontal cortex from subjects with schizophrenia at different stages of illness. Thus we aimed to validate and extend these findings. Method: We measured levels of mRNA and protein for phospholipase C beta 1 variant a and b using real-time PCR and western blot analysis, respectively, in the dorsolateral prefrontal cortex from subjects with schizophrenia, who had a short (< 7 years) or long (> 22 years) duration of illness. Results: Compared to age/sex matched controls, levels of phospholipase C beta 1 variant a and b mRNAs were decreased (−33% and −50%, respectively) in short duration schizophrenia. By contrast, only variant a mRNA was decreased (−24%) in long duration schizophrenia. There was no significant difference in the protein levels of either phospholipase C beta 1 variant in schizophrenia, irrespective of duration of illness (variant a; P = 0.84, variant b; P = 0.73). Conclusion: Our data confirm that phospholipase C beta 1 transcript levels are decreased in the dorsolateral prefrontal cortex from subjects with schizophrenia. However, the changes in levels of mRNA do not translate into a change at the level of protein. It is possible protein expression is regulated independently of mRNA and it remains to be determined whether there is a functional consequence of this change in mRNA relating to the pathophysiology of schizophrenia.

Treating Schizophrenia: Novel Targets for the Cholinergic System

Cognitive deficits in patients with schizophrenia are the biggest obstacle to achieving an independent and productive lifestyle, with these deficits being refractory to current drug treatments. Significantly, both nicotinic and muscarinic receptors (cholinoceptors) have been shown to have an important role in cognition and are therefore viewed as potential therapeutic targets for drugs designed to lessen cognitive deficits. Importantly, the demonstration that acetylcholinesterase inhibitors, which result in higher synaptic levels of acetylcholine, can reduce the cognitive deficits of schizophrenia suggested that under-stimulation of cholinoceptors could be associated with the cognitive deficits associated with this disorder. This has lead to a focus on the development of receptor agonists, partial agonists and allosteric agonists that can be used to stimulate cholinergic pathways and thus reduce the cognitive deficits of schizophrenia. In addition, muscarinic receptors have now been associated with the modulation of dopamine and may constitute an alternative target for the treatment of psychoses. Given these exciting new therapeutic initiatives, this review will outline current evidence that involves the cholinoceptors in the pathophysiology of schizophrenia and how these data can inform on approaches to more targeted treatments for the disorder.