RESUMO
The mechanisms by which common risk variants of small effect interact to contribute to complex genetic disorders are unclear. Here, we apply a genetic approach, using isogenic human induced pluripotent stem cells, to evaluate the effects of schizophrenia (SZ)-associated common variants predicted to function as SZ expression quantitative trait loci (eQTLs). By integrating CRISPR-mediated gene editing, activation and repression technologies to study one putative SZ eQTL (FURIN rs4702) and four top-ranked SZ eQTL genes (FURIN, SNAP91, TSNARE1 and CLCN3), our platform resolves pre- and postsynaptic neuronal deficits, recapitulates genotype-dependent gene expression differences and identifies convergence downstream of SZ eQTL gene perturbations. Our observations highlight the cell-type-specific effects of common variants and demonstrate a synergistic effect between SZ eQTL genes that converges on synaptic function. We propose that the links between rare and common variants implicated in psychiatric disease risk constitute a potentially generalizable phenomenon occurring more widely in complex genetic disorders.
Assuntos
Regulação da Expressão Gênica , Predisposição Genética para Doença , Células-Tronco Pluripotentes Induzidas/patologia , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas , Esquizofrenia/genética , Esquizofrenia/patologia , Sistemas CRISPR-Cas , Canais de Cloreto/antagonistas & inibidores , Canais de Cloreto/genética , Canais de Cloreto/metabolismo , Feminino , Furina/antagonistas & inibidores , Furina/genética , Furina/metabolismo , Edição de Genes , Estudo de Associação Genômica Ampla , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Masculino , Proteínas Monoméricas de Montagem de Clatrina/antagonistas & inibidores , Proteínas Monoméricas de Montagem de Clatrina/genética , Proteínas Monoméricas de Montagem de Clatrina/metabolismo , Proteínas SNARE/antagonistas & inibidores , Proteínas SNARE/genética , Proteínas SNARE/metabolismoRESUMO
CALM (clathrin assembly lymphoid myeloid leukemia protein) is a cargo-selective adaptor for the post-Golgi R-SNAREs VAMPs 2, 3, and 8, and it also regulates the size of clathrin-coated pits and vesicles at the plasma membrane. The present study has two objectives: to determine whether CALM can sort additional VAMPs, and to investigate whether VAMP sorting contributes to CALM-dependent vesicle size regulation. Using a flow cytometry-based endocytosis efficiency assay, we demonstrate that CALM is also able to sort VAMPs 4 and 7, even though they have sorting signals for other clathrin adaptors. CALM homologues are present in nearly every eukaryote, suggesting that the CALM family may have evolved as adaptors for retrieving all post-Golgi VAMPs from the plasma membrane. Using a knockdown/rescue system, we show that wild-type CALM restores normal VAMP sorting in CALM-depleted cells, but that two non-VAMP-binding mutants do not. However, when we assayed the effect of CALM depletion on coated pit morphology, using a fluorescence microscopy-based assay, we found that the two mutants were as effective as wild-type CALM. Thus, we can uncouple the sorting function of CALM from its structural role.
Assuntos
Clatrina/metabolismo , Invaginações Revestidas da Membrana Celular/metabolismo , Proteínas Monoméricas de Montagem de Clatrina/metabolismo , Proteínas R-SNARE/metabolismo , Sequência de Aminoácidos , Invaginações Revestidas da Membrana Celular/ultraestrutura , Endocitose , Expressão Gênica , Complexo de Golgi/metabolismo , Células HeLa , Humanos , Microscopia de Fluorescência , Proteínas Monoméricas de Montagem de Clatrina/antagonistas & inibidores , Proteínas Monoméricas de Montagem de Clatrina/genética , Mutação , Sinais Direcionadores de Proteínas , Transporte Proteico , Proteínas R-SNARE/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Alinhamento de Sequência , Transdução de Sinais , Vesículas Transportadoras/metabolismo , Vesículas Transportadoras/ultraestruturaRESUMO
Clathrin assembly lymphoid myeloid leukemia protein (CALM) is a clathrin assembly protein with a domain structure similar to the neuron-specific assembly protein AP180. We have previously found that CALM is expressed in neurons and present in synapses. We now report that CALM has a neuron-related function: it facilitates the endocytosis of the synaptic vesicle protein VAMP2 from the plasma membrane. Overexpression of CALM leads to the reduction of cell surface VAMP2, whereas knockdown of CALM by RNA interference results in the accumulation of surface VAMP2. The AP180 N-terminal homology (ANTH) domain of CALM is required for its effect on VAMP2 trafficking, and the ANTH domain itself acts as a dominant-negative mutant. Thus, our results reveal a role for CALM in directing VAMP2 trafficking during endocytosis.