Nevin A. Lambert

Differentiation of Human Embryonic Stem Cells to Dopaminergic Neurons in Serum‐Free Suspension Culture

The use of human embryonic stem cells (hESCs) as a source of dopaminergic neurons for Parkinsons disease cell therapy will require the development of simple and reliable cell differentiation protocols. The use of cell cocultures, added extracellular signaling factors, or transgenic approaches to drive hESC differentiation could lead to additional regulatory as well as cell production delays for these therapies. Because the neuronal cell lineage seems to require limited or no signaling for its formation, we tested the ability of hESCs to differentiate to form dopamine‐producing neurons in a simple serum‐free suspension culture system. BG01 and BG03 hESCs were differentiated as suspension aggregates, and neural progenitors and neurons were detecz after 2–4 weeks. Plated neurons responded appropriately to electrophysiological cues. This differentiation was inhibited by early exposure to bone morphogenic protein (BMP)‐4, but a pulse of BMP‐4 from days 5 to 9 caused induction of peripheral neuronal differentiation. Real‐time polymerase chain reaction and whole‐mount immunocytochemistry demonstrated the expression of multiple markers of the midbrain dopaminergic phenotype in serum‐free differentiations. Neurons expressing tyrosine hydroxylase (TH) were killed by 6‐hydroxydopamine (6‐OHDA), a neurotoxic catecholamine. Upon plating, these cells released dopamine and other catecholamines in response to K+ depolarization. Surviving TH+ neurons, derived from the cells differentiated in serum‐free suspension cultures, were detected 8 weeks after transplantation into 6‐OHDA–lesioned rat brains. This work suggests that hESCs can differentiate in simple serum‐free suspension cultures to produce the large number of cells required for transplantation studies.

GPR109A is a G-protein-coupled receptor for the bacterial fermentation product butyrate and functions as a tumor suppressor in colon.

Short-chain fatty acids, generated in colon by bacterial fermentation of dietary fiber, protect against colorectal cancer and inflammatory bowel disease. Among these bacterial metabolites, butyrate is biologically most relevant. GPR109A is a G-protein-coupled receptor for nicotinate but recognizes butyrate with low affinity. Millimolar concentrations of butyrate are needed to activate the receptor. Although concentrations of butyrate in colonic lumen are sufficient to activate the receptor maximally, there have been no reports on the expression/function of GPR109A in this tissue. Here we show that GPR109A is expressed in the lumen-facing apical membrane of colonic and intestinal epithelial cells and that the receptor recognizes butyrate as a ligand. The expression of GPR109A is silenced in colon cancer in humans, in a mouse model of intestinal/colon cancer, and in colon cancer cell lines. The tumor-associated silencing of GPR109A involves DNA methylation directly or indirectly. Reexpression of GPR109A in colon cancer cells induces apoptosis, but only in the presence of its ligands butyrate and nicotinate. Butyrate is an inhibitor of histone deacetylases, but apoptosis induced by activation of GPR109A with its ligands in colon cancer cells does not involve inhibition of histone deacetylation. The primary changes in this apoptotic process include down-regulation of Bcl-2, Bcl-xL, and cyclin D1 and up-regulation of death receptor pathway. In addition, GPR109A/butyrate suppresses nuclear factor-kappaB activation in normal and cancer colon cell lines as well as in normal mouse colon. These studies show that GPR109A mediates the tumor-suppressive effects of the bacterial fermentation product butyrate in colon.

Targeted inactivation of the sodium-calcium exchanger (Ncx1) results in the lack of a heartbeat and abnormal myofibrillar organization

Contraction of cardiac muscle is triggered by an intracellular buildup of Ca2+ during excitation‐contraction (E‐C) coupling. The Na+/Ca2+ exchanger (Ncx1) is highly expressed in cardiomyocytes and is thought to serve a housekeeping function by maintaining a low intracellular Ca2+ concentration. However, its role in E‐C coupling is controversial. To determine the precise role of Na+/Ca2+ exchange in development of the mammalian heart, we used gene targeting to delete Ncx1. Heterozygous mice are normal and fertile, whereas Ncx1‐null embryos are growth‐retarded and survive to 11.0 days postcoitum but lack a spontaneously beating heart. Moreover, normal heart morphogenesis (specification, looping, and chamber formation) occurred relatively normally within Ncx1‐null embryos. In addition, Ncx1‐nulls displayed relatively normal transient Ca2+ signals when electrically stimulated. This suggests that the Ca2+ delivery mechanism was fundamentally intact, and that Ncx1‐null cardiomyocytes can regulate intracellular Ca2+ concentrations despite the absence of Ncx1. However, ultrastructural analysis revealed that Ncx1‐null cardiomyocytes have a complete lack of organized myofibrils and Z‐lines when compared with normal littermates. These data demonstrate that Ncx‐1 is a Ca2+‐ gene that is essential for normal cardiomyocyte development and function and may serve as an animal model for functionally related human congenital heart defects.

Some G protein heterotrimers physically dissociate in living cells

Heterotrimeric G proteins mediate physiological processes ranging from phototransduction to cell migration. In the accepted model of G protein signaling, Gαβγ heterotrimers physically dissociate after activation, liberating free Gα subunits and Gβγ dimers. This model is supported by evidence obtained in vitro with purified proteins, but its relevance in vivo has been questioned. Here, we show that at least some heterotrimeric G protein isoforms physically dissociate after activation in living cells. Gα subunits extended with a transmembrane (TM) domain and cyan fluorescent protein (CFP) were immobilized in the plasma membrane by biotinylation and cross-linking with avidin. Immobile CFP-TM-Gα greatly decreased the lateral mobility of intracellular Gβ1γ2-YFP, indicating the formation of stable heterotrimers. A GTPase-deficient (constitutively active) mutant of CFP-TM-GαoA lost the ability to restrict Gβ1γ2-YFP mobility, whereas GTPase-deficient mutants of CFP-TM-Gαi3 and CFP-TM-Gαs retained this ability. Activation of cognate G protein-coupled receptors partially relieved the constraint on Gβ1γ2-YFP mobility induced by immobile CFP-TM-GαoA and CFP-TM-Gαi3 but had no effect on the constraint induced by CFP-TM-Gαs. These results demonstrate the physical dissociation of heterotrimers containing GαoA and Gαi3 subunits in living cells, supporting the subunit dissociation model of G protein signaling for these subunits. However, these results are also consistent with the suggestion that G protein heterotrimers (e.g., Gαs) may signal without physically dissociating.

Blockade of Dendritic Cell Development by Bacterial Fermentation Products Butyrate and Propionate through a Transporter (Slc5a8)-dependent Inhibition of Histone Deacetylases

Mammalian colon harbors trillions of bacteria, yet there is no undue inflammatory response by the host against these bacteria under normal conditions. The bacterial fermentation products acetate, propionate, and butyrate are believed, at least in part, to be responsible for these immunosuppressive effects. Dendritic cells play an essential role in presentation of antigens to T lymphocytes and initiation of adaptive immune responses. Here we report that butyrate and propionate block the generation of dendritic cells from bone marrow stem cells, without affecting the generation of granulocytes. This effect is dependent on the Na+-coupled monocarboxylate transporter Slc5a8, which transports butyrate and propionate into cells, and on the ability of these two bacterial metabolites to inhibit histone deacetylases. Acetate, which is also a substrate for Slc5a8 but not an inhibitor of histone deacetylases, does not affect dendritic cell development, indicating the essential role of histone deacetylase inhibition in the process. The blockade of dendritic cell development by butyrate and propionate is associated with decreased expression of the transcription factors PU.1 and RelB. Butyrate also elicits its biologic effects through its ability to activate the G-protein-coupled receptor Gpr109a, but this mechanism is not involved in butyrate-induced blockade of dendritic cell development. The participation of Slc5a8 and the non-involvement of Gpr109a in butyrate effects have been substantiated using bone marrow cells obtained from Slc5a8−/− and Gpr109a−/− mice. These findings uncover an important mechanism underlying the anti-inflammatory functions of the bacterial fermentation products butyrate and propionate.

Instability of a class a G protein-coupled receptor oligomer interface.

The quaternary structure of G protein-coupled receptors (GPCRs) can influence their trafficking and ability to transduce signals. GPCR oligomers are generally portrayed as long-lived entities, although the stability of these complexes has not been studied. Here we show that D2 dopamine receptor protomers interact transiently at a specific oligomer interface. Selective immobilization of cyan fluorescent protein-D2 receptors (C-D2Rs) in the plasma membrane failed to completely immobilize coexpressed D2-venus receptors (D2R-Vs), suggesting that the two did not form stable oligomers with each other. Oxidative cross-linking stabilized C-D2R-D2R-V oligomers such that immobilization of C-D2R also immobilized D2R-V. This stabilization required the presence in both C-D2R and D2R-V of a cysteine residue in transmembrane domain 4 (TM4), a region identified as a putative oligomer interface in these and other class A GPCRs. These results suggest that the interaction of D2 receptor protomers at TM4 is transient unless stabilized and that the quaternary structure of these receptors may thus be subject to physiological or pharmacological regulation.

CODA-RET reveals functional selectivity as a result of GPCR heteromerization.

Here we present a novel method that combines protein complementation with resonance energy transfer to study conformational changes in response to activation of a defined G protein-coupled receptor heteromer, and we apply the approach to the putative dopamine D1-D2 receptor heteromer. Remarkably, the potency of the D2 receptor (D2R) agonist R(–)-Propylnorapomorphine (NPA) to change the Gαi conformation via the D2R protomer in the D1-D2 heteromer was enhanced 10-fold relative to that observed in the D2R homomer. In contrast, the potencies of the D2R agonists dopamine and quinpirole were the same in the homomer and heteromer. Thus, we have uncovered a molecular mechanism for functional selectivity, in which a drug acts differently at a GPCR protomer depending on the identity of the second protomer that participates in forming the signaling unit, opening the door to enhanced pharmacological specificity through targeting differences between homomeric and heteromeric signaling.

Inactive-state preassembly of Gq-coupled receptors and Gq heterotrimers

G protein-coupled receptors (GPCRs) transmit signals by forming active-state complexes with heterotrimeric G proteins. It has been suggested that some GPCRs also assemble with G proteins prior to ligand-induced activation, and that inactive-state preassembly facilitates rapid and specific G protein activation. However, no mechanism of preassembly has been described, and no functional consequences of preassembly have been demonstrated. Here we show that M3 muscarinic acetylcholine receptors (M3R) form inactive-state complexes with Gq heterotrimers in intact cells. The M3R C terminus is sufficient, and a six amino-acid polybasic sequence distal to helix 8 (565KKKRRK570) is necessary for preassembly with Gq. Replacing this sequence with six alanine residues prevents preassembly, slows the rate of Gq activation, and decreases steady-state agonist sensitivity. Other Gq-coupled receptors possess similar polybasic regions and also preassemble with Gq, suggesting that these GPCRs may utilize a common preassembly mechanism to facilitate activation of Gq heterotrimers.

Three-dimensional neural constructs: a novel platform for neurophysiological investigation

Morphological and electrophysiological properties of neural cells are substantially influenced by their immediate extracellular surroundings, yet the features of this environment are difficult to mimic in vitro. Therefore, there is a tremendous need to develop a new generation of culture systems that more closely model the complexity of nervous tissue. To this end, we engineered novel electrophysiologically active 3D neural constructs composed of neurons and astrocytes within a bioactive extracellular matrix-based scaffold. Neurons within these constructs exhibited extensive 3D neurite outgrowth, expressed mature neuron-specific cytoskeletal proteins, and remained viable for several weeks. Moreover, neurons assumed complex 3D morphologies with rich neurite arborization and clear indications of network connectivity, including synaptic junctures. Furthermore, we modified whole-cell patch clamp techniques to permit electrophysiological probing of neurons deep within the 3D constructs, revealing that these neurons displayed both spontaneous and evoked electrophysiological action potentials and exhibited functional synapse formation and network properties. This is the first report of individual patch clamp recordings of neurons deep within 3D scaffolds. These tissue engineered cellular constructs provide an innovative platform for neurobiological and electrophysiological investigations, serving as an important step towards the development of more physiologically relevant neural tissue models.

Differential desensitization of responses mediated by presynaptic and postsynaptic A1 adenosine receptors.

G-protein-coupled receptors (GPCRs) often desensitize during continuous activation, but it is not known whether desensitization is influenced by subcellular location. In hippocampal neurons, activation of adenosine A1 receptors (A1Rs) or GABABreceptors on synaptic terminals inhibits neurotransmitter release, whereas activation of the same receptors on cell bodies and dendrites decreases excitability by activating inwardly rectifying potassium (GIRK) channels. Here we report that responses mediated by presynaptic A1Rs desensitize more slowly than responses mediated by postsynaptic (somatodendritic) A1Rs in cultured neurons. Agonist treatment for 2 hr has no effect on adenosine-induced presynaptic inhibition, whereas such treatment nearly abolishes adenosine-induced activation of postsynaptic GIRK channels. Agonist treatment for longer periods (>12 hr) eventually desensitizes A1R-mediated presynaptic inhibition. Presynaptic and postsynaptic responses both recover from desensitization after agonist removal, but recovery of presynaptic inhibition requires more time. Desensitization of postsynaptic responses apparently occurs at the level of the receptor, because postsynaptic G-proteins and GIRK channels appear to be fully functional. Inhibition of voltage-gated calcium channels by postsynaptic A1Rs also desensitizes rapidly, although this desensitization is less complete than is observed for activation of postsynaptic GIRK channels. Comparison of concentration–response curves for presynaptic and postsynaptic responses suggests that a receptor reserve exists for presynaptic inhibition, but that the magnitude of this reserve is insufficient to account for the absence of presynaptic desensitization after brief agonist exposure. These results suggest that agonist-induced desensitization of responses mediated by neuronal GPCRs may depend on the subcellular location of the receptors.