Association of the calcyon neuron-specific vesicular protein gene (CALY) with adolescent smoking initiation in China and California.
ABSTRACT: Although previous investigations have indicated a role for genetic factors in smoking initiation, the underlying genetic mechanisms are still unknown. In 2,339 adolescents from a Chinese Han population in the Wuhan Smoking Prevention Trial (Wuhan, China, 1998-1999), the authors explored the association of 57 genes in the dopamine pathway with smoking initiation. Using a conservative approach for declaring significance, positive findings were further examined in an independent sample of 603 Caucasian adolescents followed for up to 10 years as part of the Children's Health Study (Southern California, 1993-2009). The authors identified 1 single nucleotide polymorphism (rs2298122) in the calcyon neuron-specific vesicular protein gene (CALY) that was positively associated with smoking initiation in females (odds ratio = 2.21, 95% confidence interval: 1.49, 3.27; P = 8.4 × 10(-5)) in the Wuhan Smoking Prevention Trial cohort, and they replicated the association in females from the Children's Health Study cohort (hazard rate ratio = 2.05, 95% confidence interval: 1.27, 3.31; P = 0.003). These results suggest that the CALY gene may influence smoking initiation in adolescents, although the potential roles of underlying psychological characteristics that may be components of the smoking-initiation phenotype, such as impulsivity or novelty-seeking, remain to be explored.
Project description:The neuron-enriched, endosomal protein Calcyon (Caly) regulates endocytosis and vesicle sorting, and is important for synaptic plasticity and brain development. In the current investigation of Caly interacting proteins in brain, the microtubule retrograde motor subunit, cytoplasmic dynein 1 heavy chain (DYNC1H), and microtubule structural proteins, ? and ? tubulin, were identified as Caly associated proteins by MALDI-ToF/ToF. Direct interaction of the Caly-C terminus with dynein and tubulin was further confirmed in in vitro studies. In Cos-7 cells, mCherry-Caly moved along the microtubule network in organelles largely labeled by the late endosome marker Rab7. Expression of the dynein inhibitor CC1, produced striking alterations in Caly distribution, consistent with retrograde motors playing a prominent role in Caly localization and movement. In axons of cultured adult rat sensory neurons, Caly-positive organelles co-localized with dynein intermediate chain (DYNC1I1-isoform IC-1B) and the dynein regulator, lissencephaly 1 (LIS1), both of which co-precipitated from brain with the Caly C-terminus. Manipulation of dynein function in axons altered the motile properties of Caly indicating that Caly vesicles utilize the retrograde motor. Altogether, the current evidence for association with dynein motors raises the possibility that the endocytic and cargo sorting functions of Caly in neurons could be regulated by interaction with the microtubule transport system.
Project description:Transport of late endosomes and lysosome-related organelles (LE/LROs) in axons is essential for supplying synaptic cargoes and for removing damaged macromolecules. Defects in this system are implicated in a range of human neurodegenerative and neurodevelopmental disorders. The findings reported here identify a novel mechanism regulating LE/LRO transport based on the coordinated coupling of microtubule motors and vesicle coat proteins to the neuron-enriched, transmembrane protein calcyon (Caly). We found that the cytoplasmic C-terminus of Caly pulled down proteins involved in microtubule-dependent transport (DIC, KIF5A, p150Glued, Lis1) and organelle biogenesis (AP-1 and AP-3) from the brain. In addition, RNA interference-mediated knockdown of Caly increased the percentage of static LE/LROs labeled by LysoTracker in cultured dorsal root ganglion axons. In contrast, overexpression of Caly stimulated movement of organelles positive for LysoTracker or the AP-3 cargo GFP-PI4KII?. However, a Caly mutant (ATEA) that does not bind AP-3 was unable to pull down motor proteins from brain, and expression of the ATEA mutant failed to increase either LE/LRO flux or levels of associated dynein. Taken together, these data support the hypothesis that Caly is a multifunctional scaffolding protein that regulates axonal transport of LE/LROs by coordinately interacting with motor and vesicle coat proteins.
Project description:Calcyon regulates activity-dependent internalization of ?-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 Cal(OE) 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 Cal(OE) 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 Cal(OE) 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 Cal(OE) 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.
Project description:The link between blood pressure (BP) and cerebral function is well established. However, it is not clear whether a common mechanism could underlie the relationship between elevated BP and cognitive deficits. The expression of calcyon, a gene abundant in catecholaminergic and hypothalamic nuclei along with other forebrain regions, is increased in the brain of the spontaneously hypertensive rat (SHR) which is a widely accepted animal model of essential hypertension and attention deficit hyperactivity disorder (ADHD). Previous studies demonstrated that mice with up-regulation of calcyon in forebrain (CalOE) exhibit deficits in working memory. To date, there is no evidence directly connecting calcyon to BP regulation. Here, we investigated whether forebrain up-regulation of calcyon alters BP using radiotelemetry. We found that CalOE mice exhibited higher mean arterial pressure (MAP) compared to tTA controls. Plasma norepinephrine levels were significantly higher in CalOE mice compared to tTA controls. Silencing the transgene with doxycycline normalized BP in CalOE mice, whereas challenging the mice with 4% high salt diet for 12 days exacerbated the MAP differences between CalOE and tTA mice. High salt diet challenge also increased proteinuria and urinary thiobarbituric acid reactive substances (TBARs) in tTA and CalOE; and the increases were more prominent in CalOE mice. Taken together, our data suggest that upregulation of calcyon in forebrain could increase BP via alterations in noradrenergic transmission and increased oxidative stress during high salt challenge. Overall, this study reveals that calcyon could be a novel neural regulator of BP raising the possibility that it could play a role in the development of vascular abnormalities.
Project description:Endocytosis and vesicle trafficking are required for optimal neural transmission. Yet, little is currently known about the evolution of neuronal proteins regulating these processes. Here, we report the first phylogenetic study of NEEP21, calcyon, and P19, a family of neuronal proteins implicated in synaptic receptor endocytosis and recycling, as well as in membrane protein trafficking in the somatodendritic and axonal compartments of differentiated neurons. Database searches identified orthologs for P19 and NEEP21 in bony fish, but not urochordate or invertebrate phyla. Calcyon orthologs were only retrieved from mammalian databases and distant relatives from teleost fish. In situ localization of the P19 zebrafish ortholog, and extant progenitor of the gene family, revealed a CNS specific expression pattern. Based on non-synonymous nucleotide substitution rates, the calcyon genes appear to be under less intense negative selective pressure. Indeed, a functional group II WW domain binding motif was detected in primate and human calcyon, but not in non-primate orthologs. Sequencing of the calcyon gene from 80 human subjects revealed a non-synonymous single nucleotide polymorphism that abrogated group II WW domain protein binding. Altogether, our data indicate the NEEP21/calcyon/P19 gene family emerged, and underwent two rounds of gene duplication relatively late in metazoan evolution (but early in vertebrate evolution at the latest). As functional studies suggest NEEP21 and calcyon play related, but distinct roles in regulating vesicle trafficking at synapses, and in neurons in general, we propose the family arose in chordates to support a more diverse range of synaptic and behavioral responses.
Project description:Calcyon is a neural enriched, single transmembrane protein that interacts with clathrin light chain and stimulates clathrin assembly and clathrin-mediated endocytosis. A similar property is shared by the heterotetrameric adaptor protein (AP) complexes AP-1, AP-2, and AP-3 which recruit cargoes for insertion into clathrin coated transport vesicles. Here we report that AP medium (?) subunits interact with a YXXØ-type tyrosine motif located at residues 133-136 in the cytoplasmic domain of calcyon. Site specific mutagenesis of the critical tyrosine and bulky hydrophobic residues tyrosine 133 and methionine 136 preferentially abrogated binding of the ubiquitous and neuronal isoforms of ?3, and also impacted ?1 and ?2 binding to a lesser degree. The relevance of these interactions was explored in vivo using mice harboring null alleles of calcyon. As seen in the mutagenesis studies, calcyon deletion in mice preferentially altered the subcellular distribution of AP-3 suggesting that calcyon could regulate membrane-bound pools of AP-3 and AP-3 function. To test this hypothesis, we focused on the hilar region of hippocampus, where levels of calcyon, AP-3, and AP-3 cargoes are abundant. We analyzed brain cryosections from control and calcyon null mice for zinc transporter 3 (ZnT3), and phosphatidylinositol-4-kinase type II alpha (PI4KII?), two well-defined AP-3 cargoes. Confocal microscopy indicated that ZnT3 and PI4KII? are significantly reduced in the hippocampal mossy fibers of calcyon knock-out brain, a phenotype previously described in AP-3 deficiencies. Altogether, our data suggest that calcyon directly interacts with ?3A and ?3B, and regulates the subcellular distribution of AP-3 and the targeting of AP-3 cargoes.
Project description:Calcyon, once known for interacting directly with the dopamine D(1) receptor (D(1)DR), is implicated in various neuropsychiatric disorders including schizophrenia, bipolar disorder, and attention deficit hyperactivity disorder. Although its direct interaction with D(1)DR has been shown to be misinterpreted, it still plays important roles in D(1)DR signaling. Here, we found that calcyon interacts with the PSD-95 and subsequently forms a ternary complex with D(1)DR through PSD-95. Calcyon is phosphorylated on Ser-169 by the PKC activator phorbol 12-myristate 13-acetate or by the D(1)DR agonist SKF-81297, and its phosphorylation increases its association with PSD-95 and recruitment to the cell surface. Interestingly, the internalization of D(1)DR at the cell surface was enhanced by phorbol 12-myristate 13-acetate and SKF-81297 in the presence of calcyon, but not in the presence of its S169A phospho-deficient mutant, suggesting that the phosphorylation of calcyon and the internalization of the surface D(1)DR are tightly correlated. Our results suggest that calcyon regulates D(1)DR trafficking by forming a ternary complex with D(1)DR through PSD-95 and thus possibly linking glutamatergic and dopamine receptor signalings. This also raises the possibility that a novel ternary complex could represent a potential therapeutic target for the modulation of related neuropsychiatric disorders.
Project description:BACKGROUND: Synthetic glucocorticoids such as dexamethasone (DEX) are commonly used to promote fetal lung maturation in at-risk preterm births, but there is emerging evidence of subsequent neurobehavioral abnormalities in these children e.g. problems with inattention/hyperactivity. However, molecular pathways mediating effects of glucocorticoid overexposure on motor and cognitive development are poorly understood. METHODS: In this study with common marmoset monkeys, we investigated for neonatal and adulthood effects of antenatal DEX treatment on the expression of the corticosteroid receptors and also calcyon, a risk gene for attention-deficit/hyperactivity disorder, in the prefrontal cortex (PFC). Pregnant marmosets were exposed to DEX (5 mg/kg body weight) or vehicle during early (days 42-48) or late (days 90-96) stages of the 144-day pregnancy. RESULTS: In neonates, relative to controls, glucocorticoid receptor (GR) mRNA levels were significantly reduced after the late DEX treatment in the medial, orbital and dorsal PFC and after the early DEX treatment in the dorsal PFC. The early DEX exposure, specifically, resulted in significant reduction in calcyon mRNA expression in the medial, orbital, dorsal and lateral PFC relative to controls. Mineralocorticoid receptor (MR) mRNA levels were not significantly affected by DEX treatment. In adults, PFC GR, calcyon, and MR mRNA levels were not significantly affected by early or late prenatal DEX treatment. CONCLUSION: These findings indicate that antenatal DEX treatment could lead to short-term alterations in PFC expression of the GR and calcyon genes, with possible neurodevelopmental functional consequences.
Project description:BACKGROUND:Black adolescents see more substance use in mainstream media but seem less responsive to it than other U.S. adolescents. Black-oriented media may be more personally relevant to them. PURPOSE:To determine smoking exposure separately for black-oriented (BSME) and mainstream (MMSE) movies and assess their longitudinal relationships with smoking among black and other-race adolescents. METHODS:Two-wave (2007-2009) national cohort survey of 2341 nonsmoking (at baseline) U.S. adolescents (aged 13-19 years), analyzed in 2012. The surveys determined BMSE and MMSE based on respondents' exposure to random subsets of 50 movies from a contemporary sample of 95 black-oriented and 288 mainstream movies previously content-coded for smoking. Outcome was smoking initiation. RESULTS:Black teens had significantly more BMSE and MMSE than other teens (p's <0.001). At follow-up, 23.5% of black and 29.0% of nonblack respondents had tried smoking. Among black respondents, BMSE was related to smoking initiation at follow-up but MMSE was not. For other adolescents, both BMSE and MMSE were related to smoking initiation. CONCLUSIONS:A prospective relationship was found between exposure to smoking in movies and smoking initiation. Among black adolescents in the U.S., this was only for black-oriented movies, suggesting the importance of personal relevance of the exposures. Parents, practitioners, and producers should be aware of these potential influences of media on black teen viewers.
Project description:OBJECTIVE:Susceptibility to cigarette smoking has been used since the mid-1990s as a measure to identify youth at risk of cigarette initiation. However, it is unclear how well this measure predicts future smoking among electronic (e-)cigarette or hookah users, or among those in tobacco-friendly social environments. METHODS:We used prospective data from the Southern California Children's Health Study to evaluate the performance (sensitivity, specificity, predictive value) of a composite index assessing susceptibility to smoking, and to evaluate whether performance of the measure differed by use of e-cigarettes or hookah, or immersion in a tobacco-friendly social environment. Susceptibility to cigarette smoking was measured in 11th/12th grade (2014) among never cigarette-smokers (N=1266); follow-up data on smoking initiation were obtained approximately 16 months later. RESULTS:Overall, 16.4% of youth initiated smoking between baseline and follow-up. The sensitivity of the susceptibility to smoking index was low (46.4%), and specificity was high (79.0%). No difference in sensitivity was observed by baseline e-cigarette use; specificity was higher among never e- cigarette users. Differences in negative predictive value (NPV) and positive predictive value (PPV) were also observed by baseline e-cigarette and hookah use. Specificity was generally lower, and sensitivity was generally higher for those in tobacco-friendly social environments. CONCLUSIONS:Our findings suggest that use of the susceptibility to smoking index in cross-sectional studies of older adolescents to identify those likely to begin smoking may be inappropriate for those using alternative tobacco products (e.g., e-cigarettes or hookah).