Gelatin maleimide microgels for hematopoietic progenitor cell encapsulation.

Hematopoietic stem cells (HSCs) are the apical cells of the hematopoietic system, giving rise to cells of the blood and lymph lineages. HSCs reside primarily within bone marrow niches that contain matrix and cell-derived signals that help inform stem cell fate. Aspects of the bone marrow microenvironment have been captured in vitro by encapsulating cells within hydrogel matrices that mimic native mechanical and biochemical properties. Hydrogel microparticles, or microgels, are increasingly being used to assemble granular biomaterials for cell culture and noninvasive delivery applications. Here, we report the optimization of a gelatin maleimide hydrogel system to create monodisperse gelatin microgels via a flow-focusing microfluidic process. We report characteristic hydrogel stiffness, stability, and swelling characteristics as well as encapsulation of murine hematopoietic stem and progenitor cells, and mesenchymal stem cells within microgels. Microgels support cell viability, confirming compatibility of the microfluidic encapsulation process with these sensitive bone marrow cell populations. Overall, this work presents a microgel-based gelatin maleimide hydrogel as a foundation for future development of a multicellular artificial bone marrow culture system.

CB1 allosteric modulators and their therapeutic potential in CNS disorders.

CB1 is the most abundant GPCR found in the mammalian brain. It has garnered considerable attention as a potential therapeutic drug target. CB1 is involved in a wide range of physiological and psychiatric processes and has the potential to be targeted in a wide range of disease states. However, most of the selective and non-selective synthetic CB1 agonists and antagonists/inverse agonists developed to date are primarily used as research tools. No novel synthetic cannabinoids are currently in the clinic for use in psychiatric illness; synthetic analogues of the phytocannabinoid THC are on the market to treat nausea and vomiting caused by cancer chemotherapy, along with off-label use for pain. Novel strategies are being explored to target CB1, but with emphasis on the elimination or mitigation of the potential psychiatric adverse effects that are observed by central agonism/antagonism of CB1. New pharmacological options are being pursued that may avoid these adverse effects while preserving the potential therapeutic benefits of CB1 modulation. Allosteric modulation of CB1 is one such approach. In this review, we will summarize and critically analyze both the in vitro characterization and in vivo validation of CB1 allosteric modulators developed to date, with a focus on CNS therapeutic effects.

A novel allosteric modulator of the cannabinoid CB1 receptor ameliorates hyperdopaminergia endophenotypes in rodent models.

The endocannabinoid system (eCBs) encompasses the endocannabinoids, their synthetic and degradative enzymes, and cannabinoid (CB) receptors. The eCBs mediates inhibition of neurotransmitter release and acts as a major homeostatic system. Many aspects of the eCBs are altered in a number of psychiatric disorders including schizophrenia, which is characterized by dysregulation of dopaminergic signaling. The GluN1-Knockdown (GluN1KD) and Dopamine Transporter Knockout (DATKO) mice are models of hyperdopaminergia, which display abnormal psychosis-related behaviors, including hyperlocomotion and changes in pre-pulse inhibition (PPI). Here, we investigate the ability of a novel CB1 receptor (CB1R) allosteric modulator, ABM300, to ameliorate these dysregulated behaviors. ABM300 was characterized in vitro (receptor binding, ß-arrestin2 recruitment, ERK1/2 phosphorylation, cAMP inhibition) and in vivo (anxiety-like behaviors, cannabimimetic effects, novel environment exploratory behavior, pre-pulse inhibition, conditioned avoidance response) to assess the effects of the compound in dysregulated behaviors within the transgenic models. In vitro, ABM300 increased CB1R agonist binding but acted as an inhibitor of CB1R agonist induced signaling, including ß-arrestin2 translocation, ERK phosphorylation and cAMP inhibition. In vivo, ABM300 did not elicit anxiogenic-like or cannabimimetic effects, but it decreased novelty-induced hyperactivity, exaggerated stereotypy, and vertical exploration in both transgenic models of hyperdopaminergia, as well as normalizing PPI in DATKO mice. The data demonstrate for the first time that a CB1R allosteric modulator ameliorates the behavioral deficits in two models of increased dopamine, warranting further investigation as a potential therapeutic target in psychiatry.

Behavioral Effects of a Potential Novel TAAR1 Antagonist.

The trace amine associated receptor 1 (TAAR1) is a G-protein coupled receptor expressed in the monoaminergic regions of the brain, and represents a potential novel therapeutic target for the treatment of neurological disorders. While selective agonists for TAAR1 have been successfully identified, only one high affinity TAAR1 antagonist has been described thus far. We previously identified four potential low potency TAAR1 antagonists through an in silico screen on a TAAR1 homology model. One of the identified antagonists (compound 22) was predicted to have favorable physicochemical properties, which would allow the drug to cross the blood brain barrier. In vivo studies were therefore carried out and showed that compound 22 potentiates amphetamine- and cocaine-mediated locomotor activity. Furthermore, electrophysiology experiments demonstrated that compound 22 increased firing of dopamine neurons similar to EPPTB, the only known TAAR1 antagonist. In order to assess whether the effects of compound 22 were mediated through TAAR1, experiments were carried out on TAAR1-KO mice. The results showed that compound 22 is able to enhance amphetamine- and cocaine-mediated locomotor activity, even in TAAR1-KO mice, suggesting that the in vivo effects of this compound are not mediated by TAAR1. In collaboration with Psychoactive Drug Screening Program, we attempted to determine the targets for compound 22. Psychoactive Drug Screening Program (PDSP) results suggested several potential targets for compound 22 including, the dopamine, norepinephrine and serotonin transporters; as well as sigma 1 and 2 receptors. Our follow-up studies using heterologous cell systems showed that the dopamine transporter is not a target of compound 22. Therefore, the biological target of compound 22 mediating its psychoactive effects still remains unknown.

Pharmacological Chaperones of the Dopamine Transporter Rescue Dopamine Transporter Deficiency Syndrome Mutations in Heterologous Cells.

A number of pathological conditions have been linked to mutations in the dopamine transporter gene, including hereditary dopamine transporter deficiency syndrome (DTDS). DTDS is a rare condition that is caused by autosomal recessive loss-of-function mutations in the dopamine transporter (DAT), which often affects transporter trafficking and folding. We examined the possibility of using pharmacological chaperones of DAT to rescue DTDS mutations. After screening a set of known DAT ligands for their ability to increase DAT surface expression, we found that bupropion and ibogaine increased DAT surface expression, whereas others, including cocaine and methylphenidate, had no effect. Bupropion and ibogaine increased wild type DAT protein levels and also promoted maturation of the endoplasmic reticulum (ER)-retained DAT mutant K590A. Rescue of K590A could be blocked by inhibiting ER to Golgi transport using brefeldin A. Furthermore, knockdown of coat protein complex II (COPII) component SEC24D, which is important in the ER export of wild type DAT, also blocked the rescue effects of bupropion and ibogaine. These data suggest that bupropion and ibogaine promote maturation of DAT by acting as pharmacological chaperones in the ER. Importantly, both drugs rescue DAT maturation and functional activity of the DTDS-associated mutations A314V and R445C. Together, these results are the first demonstration of pharmacological chaperoning of DAT and suggest this may be a viable approach to increase DAT levels in DTDS and other conditions associated with reduced DAT function.

In-vivo pharmacology of Trace-Amine Associated Receptor 1.

Trace-amines (TAs) are endogenous amines that are implicated in several physiological processes including modulation of aminergic neurotransmission. These compounds exert their effect by activating a class of G protein-coupled receptors termed Trace-Amine Associated Receptors (TAARs), where TAAR1 is the only human receptor that has been shown to bind endogenous TAs. Most of the studies have focused on studying the role of TAAR1 on modulation of the dopamine transmission. These studies indicate that TAAR1 is a negative regulator of dopamine transmission making TAAR1 a novel target for neuropsychiatric disorders that arises from dopamine dysfunction such as schizophrenia. This review discusses the unique pharmacology of TAAR1 with the major focus on the physiological role of TAAR1 and its modulation of dopamine transmission.

A ß-lactamase based assay to measure surface expression of membrane proteins.

Measurement of cell surface expression is an essential part of studying membrane proteins. Traditional techniques for measuring surface expression depend on the availability of appropriate radioligands or antibodies towards extracellular epitopes of a protein of interest. The current protocol outlines the use of an assay to monitor surface expression of membrane proteins tagged with a bacterial ß-lactamase in mammalian cell lines. The use of this technique allows for quick, quantitative, sensitive, and inexpensive measurement of surface expression, with the potential for high-throughput screening.

Measurement of G protein-coupled receptor surface expression.

The quantity of G protein-coupled receptors (GPCRs) expressed on the cell surface is an important factor regulating receptor signaling. Maturation, internalization, recycling and degradation together determine the net amount of receptor surface expression. Understanding every aspect of the receptor lifecycle will facilitate the development of therapeutic applications. A number of assays for measuring the surface expression of GPCRs are currently available. This minireview summarizes the currently available assays and their suitability and usage for measuring GPCR surface expression.

A novel assay for measurement of membrane-protein surface expression using a ß-lactamase.

The trafficking of membrane proteins is dynamic and contributes to the homeostatic control of their cell surface localization and their function in signal transduction. Therefore, it is important to have sensitive techniques that allow measurement of surface expression. The current assays for such measurement are time consuming and low throughput. Here, we describe a quantitative, one-step and potentially high-throughput assay, using the ß-lactamase enzyme (ßlac) as a reporter, for measurement of surface expression of proteins. In this assay, the ßlac is fused to the extracellular portion of the plasma membrane protein of interest. To selectively measure surface expression, a cell-impermeable substrate of ßlac, nitrocefin, is used. We demonstrate the utility of the ßlac assay using well-established paradigms of internalization and molecular chaperoning, applied to two G-protein-coupled receptors and a monoamine transporter. Considering its simplicity and low cost, this assay could become a standard technique in the measurement of protein surface expression.