Clare Bergson

Dopamine receptor-interacting proteins: the Ca2+ connection in dopamine signaling

Abnormal activity of the dopamine system has been implicated in several psychiatric and neurological illnesses; however, lack of knowledge about the precise sites of dopamine dysfunction has compromised our ability to improve the efficacy and safety of dopamine-related drugs used in treatment modalities. Recent work suggests that dopamine transmission is regulated via the concerted efforts of a cohort of cytoskeletal, adaptor and signaling proteins called dopamine receptor-interacting proteins (DRIPs). The discovery that two DRIPs, calcyon and neuronal Ca(2+) sensor 1 (NCS-1), are upregulated in schizophrenia highlights the possibility that altered protein interactions and defects in Ca(2+) homeostasis might contribute to abnormalities in the brain dopamine system in neuropsychiatric diseases.

Receptor crosstalk protein, calcyon, regulates affinity state of dopamine D1 receptors

The recently cloned protein, calcyon, potentiates crosstalk between G(s)-coupled dopamine D1 receptors and heterologous G(q/11)-coupled receptors allowing dopamine D1 receptors to stimulate intracellular Ca(2+) release, in addition to cAMP production. This crosstalk also requires the participating G(q/11)-coupled receptors to be primed by their agonists. We examined the ability of calcyon and priming to regulate the affinity of dopamine D1 receptors for its ligands. Receptor binding assays were performed on HEK293 cell membrane preparations expressing dopamine D1 receptors either alone or in combination with calcyon. Co-expression of dopamine D1 receptor and calcyon affected neither the affinity of this receptor for antagonists nor the affinity of agonist binding to this receptor high and low-affinity states. However, the presence of calcyon dramatically decreased the proportion of the high-affinity dopamine D1 receptor agonist binding sites. This decrease was reversed by carbachol, which primes the receptor crosstalk by stimulating endogenous G(q/11)-coupled muscarinic receptors. Our findings suggest that calcyon regulates the ability of dopamine D1 receptors to achieve the high-affinity state for agonists, in a manner that depends on priming of receptor crosstalk.

Calcyon, a novel partner of clathrin light chain, stimulates clathrin-mediated endocytosis

In the central nervous system, clathrin-mediated endocytosis is crucial for efficient synaptic transmission. Clathrin-coated vesicle assembly and disassembly is regulated by some 30 adaptor and accessory proteins, most of which interact with clathrin heavy chain. Using the calcyon cytosolic domain as bait, we isolated clathrin light chain in a yeast two-hybrid screen. The interaction domain was mapped to the heavy chain binding domain and C-terminal regions of light chain. Further, the addition of the calcyon C terminus stimulated clathrin self-assembly in a dose-dependent fashion. Calcyon, which is a single transmembrane protein predominantly expressed in brain, localized to vesicular compartments within pre- and postsynaptic structures. There was a high degree of overlap in the distribution of LC and calcyon in neuronal dendrites, spines, and cell bodies. Co-immunoprecipitation studies further suggested an association of calcyon with the clathrin-mediated endocytic machinery. Compared with controls, HEK293 cells overexpressing calcyon exhibited significantly enhanced transferrin uptake but equivalent levels of recycling. Conversely, transferrin uptake was largely abolished in neocortical neurons obtained from mice homozygous for a calcyon null allele, whereas recycling proceeded at wild type levels. Collectively, these data indicate a role for calcyon in clathrin-mediated endocytosis in brain.

A crucial role for cAMP and protein kinase A in D1 dopamine receptor regulated intracellular calcium transients.

D1-like dopamine receptors stimulate Ca2+ transients in neurons but the effector coupling and signaling mechanisms underlying these responses have not been elucidated. Here we investigated potential mechanisms using both HEK 293 cells that stably express D1 receptors (D1HEK293) and hippocampal neurons in culture. In D1HEK293 cells, the full D1 receptor agonist SKF 81297 evoked a robust dose-dependent increase in Ca2+i following ‘priming’ of endogenous Gq/11-coupled muscarinic or purinergic receptors. The effect of SKF81297 could be mimicked by forskolin or 8-Br-cAMP. Further, cholera toxin and the cAMP-dependent protein kinase (PKA) inhibitors, KT5720 and H89, as well as thapsigargin abrogated the D1 receptor evoked Ca2+ transients. Removal of the priming agonist and treatment with the phospholipase C inhibitor U73122 also blocked the SKF81297-evoked responses. D1R agonist did not stimulate IP3 production, but pretreatment of cells with the D1R agonist potentiated Gq-linked receptor agonist mobilization of intracellular Ca2+ stores. In neurons, SKF81297 and SKF83959, a partial D1 receptor agonist, promoted Ca2+ oscillations in response to Gq/11-coupled metabotropic glutamate receptor (mGluR) stimulation. The effects of both D1R agonists on the mGluR-evoked Ca2+ responses were PKA dependent. Altogether the data suggest that dopamine D1R activation and ensuing cAMP production dynamically regulates the efficiency and timing of IP3-mediated intracellular Ca2+ store mobilization.

Elevated Intracellular Calcium Triggers Recruitment of the Receptor Cross-talk Accessory Protein Calcyon to the Plasma Membrane

Calcyon is called a “cross-talk accessory protein” because the mechanism by which it enables the typically Gs-linked D1 dopamine receptor to stimulate intracellular calcium release depends on a priming step involving heterologous Gq-linked G-protein-coupled receptor activation. The details of how priming facilitates the D1R calcium response have yet to be precisely elucidated. The present work shows that calcyon is constitutively localized both in vesicular and plasma membrane compartments within HEK293 cells. In addition, surface biotinylation and luminescence assays revealed that priming stimulates a 2-fold increase in the levels of calcyon expressed on the cell surface and that subsequent D1R activation produces further accumulation of the protein in the plasma membrane. The effects of priming and D1R agonists were blocked by nocodazole implicating microtubules in the delivery of calcyon-containing vesicles to the cell surface. Accumulation of calcyon in the plasma membrane correlated well with increased intracellular calcium levels as thapsigargin mimicked, and 2-aminoethoxydiphenylborane abrogated, the effects of priming. KN-62, an inhibitor of calcium/calmodulin-dependent protein kinase II (CaMKII) also blocked the effects of priming and D1R agonists. Furthermore, expression of constitutively active forms of the kinase bypassed the requirement for priming indicating that CaMKII is a key effector in the Ca2+ and microtubule-dependent delivery of calcyon to the cell surface.

Calcyon is necessary for activity-dependent AMPA receptor internalization and LTD in CA1 neurons of hippocampus.

Calcyon is a single transmembrane endocytic protein that regulates clathrin assembly and clathrin‐mediated endocytosis in the brain. Ultrastructural studies indicate that calcyon localizes to spines, but whether it regulates glutamate neurotransmission is not known. Here, we show that deletion of the calcyon gene in mice inhibits agonist‐stimulated endocytosis of α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptors (AMPARs), without altering basal surface levels of the GluR1 or GluR2 subunits. Whole‐cell patch‐clamp studies of hippocampal neurons in culture and CA1 synapses in slices revealed that knockout (KO) of calcyon abolishes long‐term synaptic depression (LTD), whereas mini‐analysis in slices indicated basal transmission in the hippocampus is unaffected by the deletion. Further, transfection of green fluorescent protein‐tagged calcyon rescued the ability of KO cultures to undergo LTD. In contrast, intracellular dialysis of a fusion protein containing the clathrin light‐chain‐binding domain of calcyon blocked the induction of LTD in wild‐type hippocampal slices. Taken together, the present studies involving biochemical, immunological and electrophysiological analyses raise the possibility that calcyon plays a specialized role in regulating activity‐dependent removal of synaptic AMPARs.

Up-regulation of calcyon results in locomotor hyperactivity and reduced anxiety in mice.

Gene linkage and association studies have implicated the region of chromosome 10q containing the calcyon locus with attention deficit hyperactivity disorder (ADHD), bipolar disorder, and schizophrenia susceptibility. In addition, levels of calcyon protein and transcripts are also significantly increased in postmortem tissue from schizophrenic brains. But whether altered calcyon expression might be part of the disease etiology or merely a patho-physiological side effect is not known. To begin to address this issue, we generated a transgenic mouse line (Cal(OE)) using the human calcyon cDNA in which calcyon expression is up-regulated in a number of forebrain structures including the hippocampus, prefrontal cortex (PFC), striatum, and amygdala. Compared to control littermates, the Cal(OE) mice display a range of abnormal behaviors including spontaneous hyperactivity, reduced anxiety, and/or impaired restraint (harm avoidance) that would indicate that calcyon up-regulation leads to deficits in control over behavioral output.

Establishment and maintenance of position-specific expression of the drosophila homeotic selector gene deformed

Publisher Summary The body pattern of Drosophila varies a basic segmental morphology at different positions on the anterior–posterior axis to achieve an astonishing variety of related but very different structures. At early stages of development, the morphological organization of the posterior head is similar to that of the rest of the body, but thereafter it rapidly diverges. The morphological divisions that mark the segmental boundaries of the gnathal segments are the first to appear in the visible segmentation of the Drosophila body plan, arising at about 6.5 hours of development. Among the most prominent, and the most important for the purposes of this chapter, are the mouth hooks and cirri. The mouth hooks are serrated chitinous structures that function as the jawbones and teeth of the larva. Finally, the mouth hooks develop from cells of the maxillary segment, as do the cirri, the rows of triangular papillae that flank the opening of the larval mouth.

Dopamine Receptors and Cognitive Function in Nonhuman Primates

The long-range goal of relating the neurotransmitter dopamine (DA) to specific receptors and specified cognitive processes could barely have been considered even a few years ago. The cloning of five distinct DA receptors, the development of receptor-specific ligands, the anatomical precision of immunohistochemistry and in situ hybridization, and not least, the development of sophisticated behavioral paradigms, are among the major advances that have made understanding DA’s role in cognition a reasonable goal. All of these approaches have been applied to the analysis of the anatomical and functional architecture of the DA innervation of the prefrontal cortex in macaque monkeys. The prefrontal cortex as a model system has been a focus of study because of its evolutionary expansion and differentiation in humans and its ample DA innervation in primates in general. Experimental studies in animal models, along with a plethora of neuropsychological studies in clinical populations, suggest that specifiable cognitive processes are impaired when DA transmission is altered in the prefrontal cortex. This chapter primarily reviews findings on the anatomical and receptor mechanisms that could underlie this association in nonhuman primates. New findings indicate that a major action of DA in the prefrontal association cortex is to modulate directly excitatory neurotransmission in pyramidal neurons and regulate their ability to integrate their high level sensory input. As is described in this chapter, these effects appear to be mediated in part via D1* receptors in axospinous synapses on pyramidal neurons that are engaged in maintaining information in short-term or working memory. To the extent that the neuronal architecture of prefrontal cortex is repeated in other regions of the cerebral cortex, the mechanisms elucidated may reveal general principles of receptor function in cortical circuits.

Calcyon upregulation in adolescence impairs response inhibition and working memory in adulthood.

Calcyon regulates activity-dependent internalization of α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) glutamate receptors and long-term depression of excitatory synapses. Elevated levels of calcyon are consistently observed in brains from schizophrenic patients, and the calcyon gene is associated with attention-deficit hyperactivity disorder. Executive function deficits are common to both disorders, and at least for schizophrenia, the etiology appears to involve both heritable and neurodevelopmental factors. Here, we show with calcyon-overexpressing CalOE transgenic mice that lifelong calcyon upregulation impairs executive functions including response inhibition and working memory, without producing learning and memory deficits in general. As response inhibition and working memory, as well as the underlying neural circuitry, continue to mature into early adulthood, we functionally silenced the transgene during postnatal days 28–49, a period corresponding to adolescence. Remarkably, the response inhibition and working memory deficits including perseverative behavior were absent in adult CalOE mice with the transgene silenced in adolescence. Suppressing the calcyon transgene in adulthood only partially rescued the deficits, suggesting calcyon upregulation in adolescence irreversibly alters development of neural circuits supporting mature response inhibition and working memory. Brain regional immunoblots revealed a prominent downregulation of AMPA GluR1 subunits in hippocampus and GluR2/3 subunits in hippocampus and prefrontal cortex of the CalOE mice. Silencing the transgene in adolescence prevented the decrease in hippocampal GluR1, further implicating altered fronto-hippocampal connectivity in the executive function deficits observed in the CalOE mice. Treatments that mitigate the effects of high levels of calcyon during adolescence could preempt adult deficits in executive functions in individuals at risk for serious mental illness.