Peripheral Routes to Neurodegeneration : Passing Through the Blood–Brain Barrier

A bidirectional crosstalk between peripheral players of immunity and the central nervous system (CNS) exists. Hence, blood–brain barrier (BBB) breakdown is emerging as a participant mechanism of dysregulated peripheral–CNS interplay, promoting diseases. Here, we examine the implication of BBB damage in neurodegeneration, linking it to peripheral brain-directed autoantibodies and gut–brain axis mechanisms. As BBB breakdown is a factor contributing to, or even anticipating, neuronal dysfunction(s), we here identify contemporary pharmacological strategies that could be exploited to repair the BBB in disease conditions. Developing neurovascular, add on, therapeutic strategies may lead to a more efficacious pre-clinical to clinical transition with the goal of curbing the progression of neurodegeneration.Peer reviewe

Cloning, expression and pharmacology of the mouse 5-HT4L receptor

AbstractSince most of our knowledge on pharmacological properties of brain 5-HT4 receptors have been discussed for mouse colliculi neurons, we cloned the corresponding receptor using the RT-PCR approach. As expected, the homology with the already cloned rat 5-HT4L receptor was high, revealing only 16 differences at the amino-acid level. One of the differences, proline75 in mouse, alanine75 in the already published rat sequences was not confirmed. Therefore this proline is part of the consensus sequence present in all 5-HT receptor transmembrane domain II (LVMP). Comparing the affinities of 11 agonists and five antagonists for the cloned mouse receptor (5-HT4L) expressed in LLCPK1 and the corresponding receptor in mouse colliculi shows an excellent correlation. The transfected mouse 5-HT4L receptor stimulated cAMP production. When expressed at high density, it exhibited intrinsic activity. In contrast to the previously described distribution, we found that mRNA encoding for both the short (5-HT4S) and the long form (5-HT4L) of 5-HT4 receptors are expressed in all mouse and rat brain areas

Modifying Ligand-Induced and Constitutive Signaling of the Human 5-HT4 Receptor

G protein–coupled receptors (GPCRs) signal through a limited number of G-protein pathways and play crucial roles in many biological processes. Studies of their in vivo functions have been hampered by the molecular and functional diversity of GPCRs and the paucity of ligands with specific signaling effects. To better compare the effects of activating different G-protein signaling pathways through ligand-induced or constitutive signaling, we developed a new series of RASSLs (receptors activated solely by synthetic ligands) that activate different G-protein signaling pathways. These RASSLs are based on the human 5-HT4b receptor, a GPCR with high constitutive Gs signaling and strong ligand-induced G-protein activation of the Gs and Gs/q pathways. The first receptor in this series, 5-HT4-D100A or Rs1 (RASSL serotonin 1), is not activated by its endogenous agonist, serotonin, but is selectively activated by the small synthetic molecules GR113808, GR125487, and RO110-0235. All agonists potently induced Gs signaling, but only a few (e.g., zacopride) also induced signaling via the Gq pathway. Zacopride-induced Gq signaling was enhanced by replacing the C-terminus of Rs1 with the C-terminus of the human 5-HT2C receptor. Additional point mutations (D66A and D66N) blocked constitutive Gs signaling and lowered ligand-induced Gq signaling. Replacing the third intracellular loop of Rs1 with that of human 5-HT1A conferred ligand-mediated Gi signaling. This Gi-coupled RASSL, Rs1.3, exhibited no measurable signaling to the Gs or Gq pathway. These findings show that the signaling repertoire of Rs1 can be expanded and controlled by receptor engineering and drug selection

A G(q/11)-coupled mutant histamine H(1) receptor F435A activated solely by synthetic ligands (RASSL)

Recently, G protein-coupled receptors activated solely by synthetic ligands (RASSLs) have been introduced as new tools to study G

Engineering GPCR signaling pathways with RASSLs

We are creating families of designer G-protein-coupled receptors (GPCRs) to allow for precise spatiotemporal control of GPCR signaling in vivo. These engineered GPCRs, called receptors activated solely by synthetic ligands (RASSLs), are unresponsive to endogenous ligands but can be activated by nanomolar concentrations of pharmacologically inert, drug-like small molecules. Currently, RASSLs exist for the three major GPCR signaling pathways (Gs, Gi, Gq). These new advances are reviewed here to help facilitate the use of these powerful and diverse tools

Engineering the Melanocortin-4 Receptor to Control Constitutive and Ligand-Mediated Gs Signaling In Vivo

The molecular and functional diversity of G protein–coupled receptors is essential to many physiological processes. However, this diversity presents a significant challenge to understanding the G protein–mediated signaling events that underlie a specific physiological response. To increase our understanding of these processes, we sought to gain control of the timing and specificity of Gs signaling in vivo. We used naturally occurring human mutations to develop two Gs-coupled engineered receptors that respond solely to a synthetic ligand (RASSLs). Our Gs-coupled RASSLs are based on the melanocortin-4 receptor, a centrally expressed receptor that plays an important role in the regulation of body weight. These RASSLs are not activated by the endogenous hormone α-melanocyte-stimulating hormone but respond potently to a selective synthetic ligand, tetrahydroisoquinoline. The RASSL variants reported here differ in their intrinsic basal activities, allowing the separation of the effects of basal signaling from ligand-mediated activation of the Gs pathway in vivo. These RASSLs can be used to activate Gs signaling in any tissue, but would be particularly useful for analyzing downstream events that mediate body weight regulation in mice. Our study also demonstrates the use of human genetic variation for protein engineering

Thermal Stability of the Human Immunodeficiency Virus Type 1 (HIV-1) Receptors, CD4 and CXCR4, Reconstituted in Proteoliposomes

BACKGROUND: The entry of human immunodeficiency virus (HIV-1) into host cells involves the interaction of the viral exterior envelope glycoprotein, gp120, and receptors on the target cell. The HIV-1 receptors are CD4 and one of two chemokine receptors, CCR5 or CXCR4. METHODOLOGY/PRINCIPAL FINDINGS: We created proteoliposomes that contain CD4, the primary HIV-1 receptor, and one of the coreceptors, CXCR4. Antibodies against CD4 and CXCR4 specifically bound the proteoliposomes. CXCL12, the natural ligand for CXCR4, and the small-molecule CXCR4 antagonist, AMD3100, bound the proteoliposomes with affinities close to those associated with the binding of these molecules to cells expressing CXCR4 and CD4. The HIV-1 gp120 exterior envelope glycoprotein bound tightly to proteoliposomes expressing only CD4 and, in the presence of soluble CD4, bound weakly to proteoliposomes expressing only CXCR4. The thermal stability of CD4 and CXCR4 inserted into liposomes was examined. Thermal denaturation of CXCR4 followed second-order kinetics, with an activation energy (E(a)) of 269 kJ/mol (64.3 kcal/mol) and an inactivation temperature (T(i)) of 56°C. Thermal inactivation of CD4 exhibited a reaction order of 1.3, an E(a) of 278 kJ/mol (66.5 kcal/mol), and a T(i) of 52.2°C. The second-order denaturation kinetics of CXCR4 is unusual among G protein-coupled receptors, and may result from dimeric interactions between CXCR4 molecules. CONCLUSIONS/SIGNIFICANCE: Our studies with proteoliposomes containing the native HIV-1 receptors allowed an examination of the binding of biologically important ligands and revealed the higher-order denaturation kinetics of these receptors. CD4/CXCR4-proteoliposomes may be useful for the study of virus-target cell interactions and for the identification of inhibitors

Monoaminergic and histaminergic strategies and treatments in brain diseases

The monoaminergic systems are the target of several drugs for the treatment of mood, motor and cognitive disorders as well as neurological conditions. In most cases, advances have occurred through serendipity, except for Parkinson's disease where the pathophysiology led almost immediately to the introduction of dopamine restoring agents. Extensive neuropharmacological studies first showed that the primary target of antipsychotics, antidepressants, and anxiolytic drugs were specific components of the monoaminergic systems. Later, some dramatic side effects associated with older medicines were shown to disappear with new chemical compounds targeting the origin of the therapeutic benefit more specifically. The increased knowledge regarding the function and interaction of the monoaminergic systems in the brain resulting from in vivo neurochemical and neurophysiological studies indicated new monoaminergic targets that could achieve the efficacy of the older medicines with fewer side-effects. Yet, this accumulated knowledge regarding monoamines did not produce valuable strategies for diseases where no monoaminergic drug has been shown to be effective. Here, we emphasize the new therapeutic and monoaminergic-based strategies for the treatment of psychiatric diseases. We will consider three main groups of diseases, based on the evidence of monoamines involvement (schizophrenia, depression, obesity), the identification of monoamines in the diseases processes (Parkinson's disease, addiction) and the prospect of the involvement of monoaminergic mechanisms (epilepsy, Alzheimer's disease, stroke). In most cases, the clinically available monoaminergic drugs induce widespread modifications of amine tone or excitability through neurobiological networks and exemplify the overlap between therapeutic approaches to psychiatric and neurological conditions. More recent developments that have resulted in improved drug specificity and responses will be discussed in this review.peer-reviewe