Rujuan Dai

Heat shock factor-4 (HSF-4a) is a repressor of HSF-1 mediated transcription*

Heat shock transcription factors (HSFs) regulate the expression of heat shock proteins and other molecular chaperones that are involved in cellular processes from higher order assembly to protein degradation and apoptosis. Among the human HSFs, HSF‐4 is expressed as at least two splice variants. One isoform (HSF‐4b) possesses a transcriptional activation domain, but this region is absent in the other isoform (HSF‐4a). We have recently shown that the HSF‐4a isoform represses basal transcription from heterologous promoters both in vitro and in vivo. Here we show that HSF‐4a and HSF‐4b have dramatically different effects on HSF‐1‐containing nuclear bodies, which form after heat shock. While the expression of HSF‐4b colocalizes with nuclear granules, the expression of HSF‐4a prevents their formation. In addition, there is a concurrent reduction of HSF‐1 in the nucleus, and there is reduction in its DNA binding activity and in HSE‐dependent transcription of a reporter gene. To better understand the mechanism by which HSF‐4a represses transcription, we inducibly expressed HSF‐4a in cells and found that HSF‐4a binds to the heat shock element (HSE) during attenuation of the heat shock response. Thus HSF‐4a is an active repressor of HSF‐1‐mediated transcription. This repressor function makes the HSF‐4a isoform unique within the HSF family. J. Cell. Biochem. 82: 692–703, 2001.

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.

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.